Food Environ Virol DOI 10.1007/s12560-015-9193-5
ORIGINAL PAPER
Distribution and Molecular Characterization of Hepatitis E virus in Domestic Animals and Wildlife in Croatia ˇ erni2 • Dijana Sˇkoric´2 • Tomislav Keros1 • Dragan Brnic´1 Jelena Prpic´1 • Silvija C 3 Zˇeljko Cvetnic´ • Lorena Jemersˇic´1
•
Received: 20 October 2014 / Accepted: 21 March 2015 Ó Springer Science+Business Media New York 2015
Abstract Hepatitis E is becoming a growing health concern in European countries as an increase of sporadic human cases of unknown origin has been recorded lately. Its causative agent, Hepatitis E virus (HEV), is known to have zoonotic potential and thus the role of domestic and wild animals in the chain of viral spread should be considered when investigating risk factors and the epidemiology of the disease. A comprehensive survey based on viral RNA detection was carried out in Croatia including blood, spleen and liver samples originating from 1816 different domestic and wild animals and digestive gland samples from 538 molluscs. A high HEV prevalence was detected in domestic pigs (24.5 %) and wild boars (12.3 %), whereas cattle, molluscs, ruminant and carnivore wildlife samples tested negative. Molecular characterization of both ORF1 and ORF2 genomic regions confirmed the phylogenetic clustering of the obtained sequences into genotype 3, previously reported in Europe. Furthermore, our results proved the presence of identical sequence variants in different samples, regardless of their origin, age or habitat of the host, suggesting transmission events between domestic swine, as well as between domestic swine and wild boars in the country. Moreover, a close genetic relationship of Croatian animal strains and known human HEV strains from GenBank opens the question of possible & Silvija Cˇerni [email protected] 1
Department of Virology, Croatian Veterinary Institute, Savska cesta 143, 10000 Zagreb, Croatia
2
Department of Biology, Faculty of Science, University of Zagreb, Marulic´ev trg 9a, 10000 Zagreb, Croatia
3
Department of Bacteriology and Parasitology, Croatian Veterinary Institute, Savska 143, 10000 Zagreb, Croatia
cross-species HEV transmission in Croatia, especially in the areas with an intensive swine production. Keywords HEV Animals ORF1 ORF2 Cloning Phylogenetic analysis
Introduction Hepatitis E virus (HEV, Hepeviridae family) is a causal agent of hepatitis E, the latest discovered and the least explored of the major hepatic diseases in humans (Chandra et al. 2008; Panda et al. 2007). Among hitherto identified human hepatitis viruses, it is the only one detected in animals, therefore having a zoonotic potential (Pavio et al. 2010). The infection is transmitted enterically, mainly by the consumption of contaminated food or water (Yugo and Meng 2013), and ranges from an asymptomatic to a severe course, as described in pregnant women (Navaneethan et al. 2008) and immunocompromised patients (Kamar et al. 2011; Le Coutre et al. 2009). So far, four HEV genotypes (HEV-1 to HEV-4) infecting humans have been recognised within seven genotypes of species A from genus Orthohepevirus according to the classification proposed by Smith et al. (2014). HEV-1 and HEV-2 are human specific, while two genetically more diverse genotypes HEV-3 and HEV-4 are reported from both human and animal sources. Epidemiological patterns (Van der Poel 2014; Yugo and Meng 2013) reveal that genotypes 1 and 2 are hyperendemic in developing countries, while genotypes 3 and 4 have been reported mainly in industrialised regions and are responsible for sporadic human cases. Although genotypes HEV-3 and HEV-4 have been isolated from a broad range of animals (Meng 2010; Yugo and Meng 2013), only domestic swine, wild boar and deer should be considered as
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true reservoirs of the disease (Van der Poel 2014). Hitherto persistent circulation of the virus within a closed animal population has experimentally been confirmed only for domestic swine (Andraud et al. 2013; Bouwknegt et al. 2009) in which not only faecal-oral route, but also direct contact transmission has been demonstrated (Andraud et al. 2013; Kasorndorkbua et al. 2004). Also possibly aerosol transmission has been suggested recently (Bouwknegt et al. 2009). The HEV shedding pattern, high prevalence, in many countries exceeding 60 % (Van der Poel 2014), as well as the fact that swine do not develop clinical symptoms support the assumption that domestic swine may be the main HEV reservoir and that foodborne transmission of HEV may have an important role in HEV epidemiology. A few examples of hepatitis E have been associated with the consumption of raw or undercooked food products of swine, wild boar, deer or contaminated shellfish (Said et al. 2009; Tamada et al. 2004; Tei et al. 2003; Yazaki et al. 2003). Moreover, a statistically higher seroprevalence found in pig farmers and veterinarians in comparison to the general population (Krumbholz et al. 2012) suggests that a contact exposure to swine may also be a risk factor. Even though the infection in humans is generally self-limiting, the virus may be transmitted by blood transfusion and organ transplantation, as demonstrated recently (Kamar et al. 2012). HEV RNA sequences have been detected in sewage, surface and waste waters, and have been shown to have high genetic similarity with those found in indigenous human cases and in swine and wildlife from the same geographical regions (Van der Poel 2014). The current HEV detection is based on PCR methods. HEV single-stranded RNA genome of 7.2 kb consists of three open reading frames (Okamoto 2007; Tam et al. 1991), from which ORF1, encoding non-structural proteins, and ORF2, encoding a capsid protein, are mainly used for inferring the virus phylogeny. The recent serological study in Croatia confirmed antiHEV antibodies in 10 % of 439 tested patients with liver damage in which the absence of acute viral hepatitis A, B or C markers was confirmed (Dakovic Rode et al. 2013) demonstrating the possibility of zoonotic HEV transmission in the country. This hypothesis was further corroborated by the first HEV human case described in Croatia (Cˇivljak et al. 2013) proven to be autochthonous. To determine the potential sources of HEV in the country, we have performed a comprehensive epidemiological survey based on HEV RNA detection. HEV presence was tested in 2354 domestic and wild animals of a different age and sex, and representatives of ten animal species found in Croatia including molluscs (mussels and oysters) as indicators of a possible sea contamination. In order to determine the phylogenetic relationship of Croatian HEV strains with those already published,
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selected ORF1 and ORF2 amplicons were sequenced. Due to the complex RNA virus population structure (Ojosnegros et al. 2011) and in order to gain better epidemiological insight of HEV transmission routes, all amplicons were cloned prior to further analyses.
Methods Samples Samples of different symptomless animals bred in farms (domestic) or hunted during the hunting season (wild) were collected during 2009 and 2010 from different locations covering all Croatian counties (Fig. 1). In total, 2354 samples were analysed: 536 wild boars (Sus scrofa L.), 848 domestic pigs (Sus scrofa domestica), 32 cattle (Bos taurus), 40 roe deer (Capreolus capreolus), 280 red deer (Cervus elaphus), 12 muflons (Ovis musimon), 50 foxes (Vulpes vulpes), 10 martens (Martes martes), 8 ferrets (Mustela putorius), 252 mussels (Mytilus galloprovincialis) and 286 oysters (Ostrea edulis). Swine samples (all categories of domestic pigs and wild boars) were collected through a national classical swine fever monitoring programme, while other samples (cattle, roe and red deer, muflons, foxes, martens, ferrets and molluscs) were collected for diagnostics of different virus diseases routinely performed in Croatian Veterinary Institute. Digestive gland samples were collected from mussels and oysters, while blood, spleen and liver samples were collected from all other investigated animal species. Sera were separated from cellular elements by centrifuging coagulated blood (the blood clots were rimmed with a sterile glass stick to facilitate separation) for 15 min at 10009g. An amount of 140 ll of each serum sample was used for viral RNA purification using the QIAamp viral RNA extraction kit (Qiagen) according to the manufacturer’s instructions. Approximately, 0.1 g of spleen, liver and digestive gland samples were homogenised and diluted in 1 ml of phosphate-buffered saline (PBS; pH 7.4). The homogenised samples were then vortexed for 1 min and centrifuged for 15 min at 10009g. An aliquot (140 ll) of each sample supernatant was used for the viral RNA isolation by QIAamp viral RNA extraction kit (Qiagen) according to the manufacturer’s protocol. The RNA samples isolated from blood, spleen and liver of each sample, excluding molluscs, were pooled, and RNA was stored at -80 °C until needed. HEV RNA Detection The amplification of HEV ORF1 and ORF2 fragments was adapted from previously published methodology (Huang
Food Environ Virol Fig. 1 Geographical origin of the examined animal samples originated from different Croatian counties. Negative samples are marked with empty and positive samples with filled shapes
et al. 2002; Van der Poel et al. 2001). Six microlitres of RNA extracts was used for reverse transcription by SuperScript III reverse transcriptase using random hexamers (Invitrogen). Two pairs of HEV primers were used for direct and nested PCR amplification of 287-nucleotidelong product of ORF1 (ORF1-s1/ORF1-a1—direct and ORF1-s2/ORF1-a2—nested) (Wang et al. 1999) and 348-nucleotide-long product of ORF2 region (ORF2-3156/ ORF2-3157—direct and ORF2-3158/ORF2-3159—nested) (Huang et al. 2002). The reaction mixtures of 50 ll containing 6 ll of cDNA were prepared following the manufacturer’s instructions (PlatinumÒ Taq DNA polymerase, Invitrogen). The direct and nested PCR parameters for the optimised amplification assay were mutually identical. The amplification procedure for ORF1 region was as follows: denaturation at 94 °C for 3 min; incubation at 94 °C for 45 s, 55 °C for 45 s, and 72 °C for 45 s (35 cycles); and incubation at 72 °C for 20 min. ORF2 region amplification procedure was denaturation at 94 °C for 3 min; incubation at 94 °C for 3 min, 50 °C for 1 min, and 72 °C for 1 min (30 cycles); and incubation at 72 °C for 20 min. PCR products were
separated by agarose gel electrophoresis in 1.5 % agarose gel stained with ethidium bromide and visualised by UV transillumination. All standard precautions were followed to prevent PCR contamination. For the verification of the results, no template, as well as positive control, was included in each amplification experiment. As a positive control, HEV strain that had previously been characterised was used. HEV Genotyping To determine the phylogenetic clustering of Croatian HEV strains, eleven positive swine samples, randomly selected representatives of different counties, were selected for further genotyping (Figs. 3, 4). Different genomic variants present in RNA virus populations were separated by TA cloning of nested PCR amplicons into the pTZ57R/T vector (Fermentas) as described by the manufacturer. The transformation of competent Escherichia coli XL-1 Blue cells was done using commercial InsTAcloneTM PCR cloning kit (Fermentas). Transformed cell colonies were selected by a-complementation, and the
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presence of the insert was confirmed by PCR using the same primer pairs and nested PCR protocols as previously described. Three clones per sample transformed with ORF2 as well as three clones per sample transformed with ORF1 amplicons were chosen randomly. Plasmids were purified using a PureLinkTM Quick Plasmid Miniprep Kit (Invitrogen), and inserts were sequenced in both directions (Macrogen) using a pair of M13-pUC universal primers. To determine the phylogenetic clustering of the obtained sequences, the prototype ORF1 and ORF2 sequences, representing all proposed Orthohepevirus A genotypes (Smith et al. 2014) were retrieved from GenBank. Also, similar sequences obtained using BLAST algorithm were included in the analysis. Sequences were aligned using ClustalX, version 2.0 (Thompson et al. 1997) and analysed using MEGA 5 (Tamura et al. 2011). Phylogenetic trees were generated using the neighbour-joining method applying Tamura-Nei evolutionary model. The tree topology was evaluated by bootstrap analysis based on 1000 repetitions. Statistical Analysis For the comparison of categorical variables between groups, v2 test was used. A p value of B0.05 was considered statistically significant.
Results Virus Detection Both HEV ORF1 and ORF2 amplicons were detected from pooled samples in 24.5 % of tested domestic pigs and in 12.3 % of tested wild boars. All positive animals originated from the north and north-east Croatian regions including 7 (Virovitica Podravina, Osijek Baranja, Vukovar Srijem, Bjelovar Bilogora, Brod Posavina, Sisak Moslavina and Koprivnica Krizˇevci) and 4 (Virovitica Podravina, Vukovar Srijem, Brod Posavina, Sisak Moslavina) counties, respectively (Fig. 1). The HEV prevalence detected in the counties where positive samples were detected ranged from 13.3 to 57.9 % for domestic pigs and from 20 to 31.8 % for tested wild boars (Table 1). HEV was found in all age categories, regardless of sex or breeding conditions. However, most of the positive samples (Fig. 2) were found within domestic pigs up to 6 months of age and wild boars up to 1 year of age. Even though the difference of HEV incidence among age groups of domestic pigs and wild boars was highly significant, the geographical distribution was unrelated to the incidence of positive samples from both groups (p [ 0.05 and p = 0.65, respectively). HEV
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RNA was not detected in other animal and molluscs samples. In each amplification experiment, clear amplification signal was detected when using positive control and no amplification when using no-template control. Sequence Analysis The sequence analysis of ORF1 and ORF2 clones confirmed the coexistence of different genomic variants in all tested samples but one (CRO OB F). Obtained sequences (i.e., CRO SM W b) were marked by country of origin (CRO), twoletter acronyms representing county, mark indicating the type of habitat (F-domestic farm animals, D-domestic backyard animals, W-wild boars) and small letter (a–c) for a clone number. All sequences displaying nucleotide differences obtained in this work were submitted to GenBank excluding primer regions. Accession numbers are as follows: KJ850446–KJ850476, KF366506–KF366510, KF366514– KF366518 and KF366520–KF366524. The phylogenetic relationship analyses based on both ORF1 and ORF2 amplicons confirmed that all sequences obtained from domestic pigs and wild boars clustered into genotype 3 (Figs. 3, 4) of Orthohepevirus A according to the classification proposed by Smith et al. (2014). Based on both ORF1 and ORF2 phylogenies supported by the high bootstrap values, the sequences of the majority of Croatian strains clustered into one of two subgroups: cluster CRO A (CRO VS W, CRO VS D, CRO OB F, CRO BB D, CRO SM W, CRO SM D) and cluster CRO B (CRO BP W, CRO BP D). Also, based on both ORF1 and ORF2 phylogenies, in sample CRO VP F, sequences clustering into both subgroups (CRO A and CRO B) were detected. The two inconsistencies were found in samples CRO VS F and CRO KK D. In samples CRO VS F, based on ORF1 phylogeny, the sequences clustered into subgroup CRO A, while based on ORF2 phylogeny, sequences clustering into both subgroups were detected. However, in sample CRO KK D based on ORF1 phylogeny, the variants clustered into subgroup CRO B, while based on ORF2 phylogeny, they clustered into subgroup CRO A. The comparison of Croatian ORF1 sequences (Fig. 3) with reference database sequences showed the highest genetic similarities with Hungarian wild boar (97.5 %; EU718647), German human (97.5 %; FN994999), Serbian swine (93.1 %; HM483380) and USA human (91.8 %; JN837481) strains. Croatian ORF2 sequences (Fig. 4) displayed the highest genetic similarity with Japanese human (95.9 %; AB089824), USA swine (94.4 %; AF466682) and Hungarian human (94.4 %, EF530669) HEV strains. By comparing sequences of different Croatian strains mutually, 100 % nucleotide identity was found in wild and domestic animals (grown on farms and backyards) from the same county (Vukovar Srijem) as well as from wild and
Food Environ Virol Table 1 The list of domestic and wild animals from different Croatian counties tested for the presence of HEV RNA County Virovitica Podravina (VP) Osijek Baranja (OB)
Vukovar Srijem (VS)
Bjelovar Bilogora (BB)
Brod Posavina (BP)
Sisak Moslavina (SM)
Koprivnica Krizˇevci (KK)
Med¯imurje (M)
Varazˇdin (V) Krapina Zagorje (KZ)
Zagreb (ZG)
Pozˇega Slavonija (PS)
Karlovac (K)
Primorje Gorski kotar (PG)
Species (habitat)
Number of positive results/tested animals
Percentage of positive samples (%)
Pig (farm)
38/115
33.0
Wild boar
9/30
30.0
Pig (farm)
70/336
20.8
Wild boar
0/84
0
Cattle
0/10
0
Roe deer, Red deer
0/7, 0/40
0
Pig (farm)
13/50
26.0
Pig (backyard)
20/58
34.5
Wild boar
6/30
20.0
Roe deer, Red deer, Muflon
0/10, 0/50, 0/4
0
Pig (backyard)
2/15
13.3
Cattle
0/10
0
Red deer, Muflon
0/80, 0/3
0
Pig (backyard)
7/18
38.8
Wild boar
21/66
31.8
Roe deer, Red deer
0/10, 0/70
0
Pig (backyard)
55/95
57.9
Wild boar
30/95
31.6
Fox
0/9
0
Pig (backyard)
3/15
20.0
Wild boar
0/33
0
Cattle
0/10
0
Pig (backyard)
0/10
0
Wild boar
0/21
0
Fox
0/10
0
Pig (backyard)
0/10
0
Fox, Ferret, Marten
0/5, 0/8, 0/5
0
Pig (backyard)
0/14
0
Wild boar
0/19
0
Roe deer
0/5
0
Pig (farm)
0/55
0
Pig (backyard)
0/10
0
Wild boar
0/20
0
Fox, Marten, Roe deer
0/10, 0/5, 0/8
0
Pig (backyard)
0/18
0
Wild boar
0/42
0
Fox
0/6
0
Pig (backyard)
0/15
0
Wild boar
0/30
0
Fox
0/10
0
Pig (backyard)
0/8
0
Wild boar
0/23
0 0
Cattle
0/12
Istria (I)
Red deer, Muflon, Mussel, Oyster
0/40, 0/5, 0/51, 0/97
0
Lika Senj (LS)
Wild boar
0/43
0
Zadar (ZD) Sˇibenik Knin (SˇK)
Mussel, Oyster
0/48, 0/96
0
Mussel
0/67
0
Split Dalmacija (SD)
Pig (backyard)
0/6
0
Dubrovnik Neretva (DN)
Mussel, Oyster
0/86, 0/93
0
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% of positive samples
Food Environ Virol
30,00 25,00 20,00 15,00 10,00 5,00 0,00
age groups domesc pigs
wild boars
Fig. 2 The prevalence of HEV infection in wild boars and domestic pigs detected by nested RT-PCR and categorised in different age groups. Wild boars category and the number of tested samples: shoats (\1 year, 187), yearlings (1–2 years, 176), adults ([2 years, 173). Domestic pigs category and the number of tested samples: sucklings
(1–4 weeks, 119), weanlings (5–10 weeks, 110), fatlings ([10 weeks, 256) and domestic pigs older than 6 months (boars 108; gilts 121; sows 139). For comparison of categorical variables between groups, v2 test was used
domestic animals (grown in backyards) from the same county (Brod Posavina and Sisak Moslavina; Figs. 3, 4— diamond markings). Moreover, 100 % nucleotide identity was found in all three categories of animals originating from different counties: (i) in farm animals from counties Virovitica Podravina, Vukovar Srijem and Osijek Baranja; (ii) in backyard animals from counties Vukovar Srijem, Bjelovar Bilogora and Koprivnica Krizˇevci, as well as from counties Virovitica Podravina and Koprivnica Krizˇevci; and (iii) in wild boars from counties Sisak Moslavina and Vukovar Srijem.
zoonotic HEV genotypes among wild animals. As the HEV monitoring programme has not been established in the country yet, samples used in this study were collected through a national classical swine fever monitoring programme or other programmes for monitoring common animal viruses. According to our RT-PCR results, HEV RNA was detected in all age groups of domestic pigs and wild boars sampled in Croatia. The correlation of both positive and negative results obtained using ORF1 and ORF2 primer sets was 100 % for all tested samples. The high sensitivity of the RT-PCR assay used was previously confirmed by Huang et al. (2002) and Wang et al. (1999) testing the sensitivity of the assay using the same ORF2 and ORF1 primer sets, respectively. Interestingly, HEV RNA was not confirmed in any other domestic or wild animal included in this study. Part of the explanations could be that the primer sets used are not specific for HEV genotypes belonging to more distantly related HEV species -B, -C or -D, (Smith et al. 2014), amongst which HEV variants from wild animals, such as ferret and mink, were lately identified. Nevertheless, the zoonotic potential of these strains has not been reported, and therefore their detection was not our priority. On the other hand, our sequence analysis indicates that at least one of the primer sets used would amplify HEV variants from all genotypes described within genus Orthohepevirus A, four of which have zoonotic potential. The average HEV prevalence in swine samples appeared to be quite high; 24.5 % of domestic swine and 12.3 % of wild boars proved to be HEV RNA positive with the
Discussion In the last decade, a few sporadic cases of foodborne HEV infection through the consumption of uncooked contaminated animal products and shellfish (Koizumi et al. 2004; Said et al. 2009; Tamada et al. 2004; Tei et al. 2003), but also epidemic outbreaks due to contaminated water or water supplies (Ceylan et al. 2003; El-Esnawy 2000; Rodriguez-Lazaro et al. 2012), have been reported. However, the cause of infection in many autochthonous sporadic cases reported in low or non-endemic countries mostly remain unidentified. In our study, samples from different wild and domestic animals, as well as molluscs, all potential HEV reservoirs, used for food were examined for the presence of HEV RNA. Furthermore, muflons, foxes, martens and ferrets were included in the analysis with the aim to explore their possible role in the spread of the
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Food Environ Virol CRO VS W c
Fig. 3 Neighbour-joining phylogenetic tree obtained by the analysis of HEV ORF1 sequences (242 nt) of genomic variants from Croatian wild boar and domestic pig HEV samples. Genotype reference sequences were adopted from Smith et al. (2014). As an outgroup, HEV B prototype sequence (Smith et al. 2014) was used. Identical sequences found in the different animal groups from the same county were marked (diamond symbol). Bootstrap values are presented next to tree nodes. The bar represents 0.005 nucleotide substitution per site
CRO VS D a CRO OB F a 14
CRO VS D b CRO VS W a
99
CRO VP F a CRO BB D
22
CRO OB F b 6
HM483380 CRO SM D a JN837481
17
cluster CRO A
AF082843
39
CRO SM D b
71
3
44 CRO SM W b 99
CRO VS W b
77
CRO SM W a CRO SM D c
16
39
HM483383 EU718646
23 26
46
AB108666 EU718647 38
AY115488
GENOTYPE 3
AB301710
80
AB114179
57
AF336013
40 56
FJ705359
95
AB290312
26
AF455784 AF195064
48
HM052802
19
JF431046 EU718650
22
JN415693 68
AF110389 95
FN994999
48
64 CRO VS F b
CRO KK D a
93
19
CRO BP W CRO VS F a
91
CRO BP D a
cluster CRO B
61 CRO KK D c
CRO VP F b
28
6 CRO BP D b 31
CRO KK D b
61 76
AB573435
GENOTYPE 5
AB602441
GENOTYPE 6
KJ496143
GENOTYPE 7
AJ272108
GENOTYPE 4
M74506 35
GENOTYPE 2
M73218
GENOTYPE 1
AY535004
HEV B (outgroup)
0.05
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Food Environ Virol CRO VP F b
Fig. 4 Neighbour-joining phylogenetic tree obtained by the analysis of HEV ORF2 sequences (301 nt) of genomic variants from Croatian wild boar and domestic pig HEV samples. Genotype reference sequences were adopted from Smith et al. (2014). As an outgroup, HEV B prototype sequence (Smith et al. 2014) was used. Identical sequences found in the different animal groups from the same county were marked (diamond symbol). Bootstrap values are presented next to tree nodes. The bar represents 0.05 nucleotide substitution per site
CRO KK D CRO BB D 64
CRO OB F CRO VS D a
100
CRO VS F b CRO VS W
47
cluster CRO A
CRO VS D b CRO SM W a CRO SM D a CRO SM W b
100
41
46 CRO SM D b 44
CRO SM D c
AB089824
74
AB094270
15
AF466682
56
25
AF082843 AB290312
41
GENOTYPE 3
FJ705359
20
AF336290
90 45
AB301710 AF336292 AF455784
49
AB094231
59
EU684329
84
EF530669
68
CRO VS F c
92 9
CRO BP W a
100
CRO BP D a
68 20 66 24
CRO BP W b CRO VP F a
cluster CRO B
CRO VS F a
12 CRO BP D c 16 CRO VP F c
0
21 CRO BP D b
AB602441
GENOTYPE 6
KJ496143
13 17
AB573435 AJ272108
71
GENOTYPE 5 GENOTYPE 4
M74506
33
GENOTYPE 7
M73218 AY535004
GENOTYPE 2 GENOTYPE 1 HEV B (outgroup)
0.05
highest prevalence (Table 1) reported in two neighbouring counties Sisak Moslavina and Brod Posavina reaching 57.9 % in domestic swine and 31.8 % in wild boar populations. Since the sample set available for testing was not the representative of the distribution of domestic pigs and wild boar in different Croatian counties, in some cases, the results might have been affected by a small sample size. A previous study in Croatia (Lipej et al. 2013), even in a small amount of tested samples, confirmed an extremely high seroprevalence of HEV-specific antibodies in the domestic swine population (91.7 %). Therefore, a high
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prevalence of the viral RNA was expected, even though the viral RNA prevalence reported by Lipej and collaborators (2013) was relatively low (13.3 % for serum and 8.1 % for bile samples, respectively). A high HEV prevalence is not uncommon in many European countries (Berto et al. 2012; Ferna´ndez-Barredo et al. 2007; Martelli et al. 2008; Reuter et al. 2009; Rutjes et al. 2007; Schielke et al. 2009), among which the prevalence reported in Hungary (Reuter et al. 2009) and Spain (Ferna´ndez-Barredo et al. 2007) were the most similar to the one reported in domestic swine in Croatia. However, the HEV prevalence reported in wild
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boars was the most similar to those reported in Germany (Schielke et al. 2009) and Hungary (Reuter et al. 2009). All positive samples were geographically located in the north and north-east part of the country, with positive domestic pigs found in seven counties and wild boars found in four of them (Fig. 1). In wild boars, the incidence distribution found by age is consistent with the dynamics described for HEV infection in Germany (Schielke et al. 2009). The reason why we have not detected the virus in the yearlings category (Fig. 2) might be relatively small number of tested animals from that age group. As described by Pavio et al. (2010), the peak of viremia in domestic swine was usually observed at 15 weeks of age corresponding to our fatlings category (Fig. 2). However, our data show the highest prevalence of HEV infection in the age class of 1–4 weeks (Fig. 2 sucklings). HEV infections in very young piglets have previously been reported (de Deus et al. 2008), but the explanation is still unclear. Possible reasons could be the horizontal sow-to-piglet transmission, which in our case is corroborated by the high HEV prevalence in sows category, or transplacental transmission as which so far has not been confirmed experimentally in swine (de Deus et al. 2008). Since positive animals have been found amongst all age groups of animals used for human consumption, this finding raises further public health concerns. The sequence analysis of cloned ORF1 and ORF2 amplicons, even in a small number of clones tested, revealed the complex HEV populations consisting of different genomic variants in all but one analysed sample, which is in accordance with quasispecies nature of RNA virus populations (Domingo et al. 2012). The phylogeny of both ORF1 and ORF2 regions (Figs. 3, 4) confirmed the clustering of Croatian strains into genotype 3, that is, excluding a few sporadic findings of genotype 4, a common HEV genotype found in Europe (Riveiro-Barciela et al. 2013). Although the latest publications (Olivera-Filho et al. 2013; Smith et al. 2014) do not support firm subgenotype classification, based on the one published by Lu et al. (2006) and Steyer et al. (2011), Croatian cluster CRO A would be classified as subgenotype 3a, whilst Croatian cluster CRO B would be classified as subgenotype 3e. The additional ORF2 sequence variants belonging to different clusters obtained in sample CRO VS F could simply be explained by a small number of clones analysed in this study. However, the explanation of the complete inconsistency found in the clustering of ORF1 and ORF2 sequence variants of sample CRO KK D could be the recombination, which, due to the complex within-host virus populations of RNA viruses, is not a rare event. Interestingly, in three Croatian counties (Virovitica Podravina, Vukovar Srijem and Koprivnica Krizˇevci), animals co-infected with virus variants from two subgroups were found. However, the pattern of
clustering into subgroups could not be linked with geographical distribution and was not species specific since both subgenotypes were detected in domestic swine, from both farms and backyards, as well as from wild boars. Even so, the results of mutual comparison of Croatian HEV sequences exposed a few possible HEV transmission events within the country, while identical sequence variants found in farm pigs from three neighbouring counties (Osijek Baranja, Vukovar Srijem and Virovitica Podravina) may be related to the introduction of breeding animals from the same, previously infected source; the finding of identical sequence variants in farm and backyard swine, as well as in wild boars in the same county (Vukovar Srijem), is most probably the result of environmental transmission. This hypothesis seems to be applicable in two more counties (Brod Posavina and Sisak Moslavina) where identical sequence variants were found in domestic swine grown in backyards and in wild boars. Moreover, identical sequence variants were found in tested animals originating from different counties. They were found in backyard pigs from neighbouring counties (Bjelovar Bilogora and Koprivnica Krizˇevci; Virovitica Podravina and Koprivnica Krizˇevci), but also in wild boars from non-neighbouring counties (Sisak Moslavina and Vukovar Srijem). The most likely scenario is that the virus was transmitted from a farm to domestic pigs and further on to wild boars due to strict bio-security measures implemented on farms that would prevent a vice versa course of infection. The accurate source of infection in domestic pigs is difficult to define due to an intensive import from different European countries in the past decades. However, the comparison of Croatian HEV sequences with those reported in GenBank indicates the highest sequence similarity of Croatian strains with those from Serbia and Hungary, which are bordering countries. To understand this complex epidemiological scenario, more detailed molecular epidemiology studies encompassing a higher number of samples from each geographical location and a higher number of clones analysed per sample would be necessary. The comparison of Croatian HEV sequences with the reference sequences from GenBank revealed a surprising finding that a high sequence similarity was reported among Croatian swine sequences and those from human HEV strains originating from Germany, Japan, Hungary and USA. This strongly suggests the possible zoonotic transmission of Croatian HEV variants described here, especially after the first autochthonous human HEV in Croatia was diagnosed (Cˇivljak et al. 2013). Moreover, a recent serological study (Dakovic Rode et al. 2013) confirmed anti-HEV antibodies in even 10 % of 439 tested patients with liver damage and the absence of acute viral hepatitis A, B or C markers in Croatia. To complete the general epidemiological picture of HEV transmission in Croatia,
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thorough analyses of surface waters from the vicinity of large industrial and small backyard pig holdings, positioned mainly in the continental part of the country, would provide reliable information regarding the HEV presence in the environment and probable routes of environmental virus transmission. Moreover, further studies on the role of domestic pigs and wild animals in HEV transmission chain are necessary to develop measures for prevention of zoonotic and environmental transmission to humans and other animal species. The fact that HEV RNA was not detected in other domestic or wild animals cannot, however, exclude them to be involved in the virus shedding, since so far only pigs have been recognised as truth HEV reservoirs. Nevertheless, the presence of HEV within the wild boar population may allow further spread of disease to other wild animals and distant locations increasing the risk of possible human infection. In conclusion, our results confirm that the genotype 3 of HEV RNA was detected in all major swine growing regions in Croatia indicating that domestic pigs and wild boars may be important reservoirs of HEV in the country presenting a significant risk for HEV infection in humans. Acknowledgments We thank Ivana Prajdic´ and Marin Jezˇic´ for their technical assistance. This study was supported by the Croatian Ministry of Science, Education, and Sports (Project No. 048-0481186-1183).
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novel genotype 3 lineage. Infection, Genetics and Evolution, 11(7), 1732–1737. Tam, A. W., Smith, M. M., Guerra, M. E., Huang, C. C., Bradley, D. W., Fry, K. E., & Reyes, G. R. (1991). Hepatitis E virus (HEV): molecular cloning and sequencing of the full-length viral genome. Virology, 185(1), 120–131. Tamada, Y., Yano, K., Yatsuhashi, H., Inoue, O., Mawatari, F., & Ishibashi, H. (2004). Consumption of wild boar linked to cases of hepatitis E. Journal of Hepatology, 40(5), 869–870. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28(10), 2731–2739. Tei, S., Kitajima, N., Takahashi, K., & Mishiro, S. (2003). Zoonotic transmission of hepatitis E virus from deer to human beings. Lancet, 362(9381), 371–373. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., & Higgins, D. G. (1997). The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25(24), 4876–4882. Van der Poel, W. H. (2014). Food and environmental routes of hepatitis E virus transmission. Current Opinion in Virology, 4C, 91–96. Van der Poel, W. H., Verschoor, F., van der Heide, R., Herrera, M. I., Vivo, A., Kooreman, M., & de Roda Husman, A. M. (2001). Hepatitis E virus sequences in swine related to sequences in humans, The Netherlands. Emerging Infectious Diseases, 7(6), 970–976. Wang, Y., Ling, R., Erker, J. C., Zhang, H., Li, H., Desai, S., et al. (1999). A divergent genotype of hepatitis E virus in Chinese patients with acute hepatitis. Journal of General Virology, 80(1), 169–177. Yazaki, Y., Mizuo, H., Takahashi, M., Nishizawa, T., Sasaki, N., Gotanda, Y., & Okamoto, H. (2003). Sporadic acute or fulminant hepatitis E in Hokkaido, Japan, may be food-borne, as suggested by the presence of hepatitis E virus in pig liver as food. The Journal of General Virology, 84(9), 2351–2357. Yugo, D. M., & Meng, X.-J. (2013). Hepatitis E virus: foodborne, waterborne and zoonotic transmission. International Journal of Environmental Research and Public Health, 10(10), 4507–4533.
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ORIGINAL PAPER
Distribution and Molecular Characterization of Hepatitis E virus in Domestic Animals and Wildlife in Croatia ˇ erni2 • Dijana Sˇkoric´2 • Tomislav Keros1 • Dragan Brnic´1 Jelena Prpic´1 • Silvija C 3 Zˇeljko Cvetnic´ • Lorena Jemersˇic´1
•
Received: 20 October 2014 / Accepted: 21 March 2015 Ó Springer Science+Business Media New York 2015
Abstract Hepatitis E is becoming a growing health concern in European countries as an increase of sporadic human cases of unknown origin has been recorded lately. Its causative agent, Hepatitis E virus (HEV), is known to have zoonotic potential and thus the role of domestic and wild animals in the chain of viral spread should be considered when investigating risk factors and the epidemiology of the disease. A comprehensive survey based on viral RNA detection was carried out in Croatia including blood, spleen and liver samples originating from 1816 different domestic and wild animals and digestive gland samples from 538 molluscs. A high HEV prevalence was detected in domestic pigs (24.5 %) and wild boars (12.3 %), whereas cattle, molluscs, ruminant and carnivore wildlife samples tested negative. Molecular characterization of both ORF1 and ORF2 genomic regions confirmed the phylogenetic clustering of the obtained sequences into genotype 3, previously reported in Europe. Furthermore, our results proved the presence of identical sequence variants in different samples, regardless of their origin, age or habitat of the host, suggesting transmission events between domestic swine, as well as between domestic swine and wild boars in the country. Moreover, a close genetic relationship of Croatian animal strains and known human HEV strains from GenBank opens the question of possible & Silvija Cˇerni [email protected] 1
Department of Virology, Croatian Veterinary Institute, Savska cesta 143, 10000 Zagreb, Croatia
2
Department of Biology, Faculty of Science, University of Zagreb, Marulic´ev trg 9a, 10000 Zagreb, Croatia
3
Department of Bacteriology and Parasitology, Croatian Veterinary Institute, Savska 143, 10000 Zagreb, Croatia
cross-species HEV transmission in Croatia, especially in the areas with an intensive swine production. Keywords HEV Animals ORF1 ORF2 Cloning Phylogenetic analysis
Introduction Hepatitis E virus (HEV, Hepeviridae family) is a causal agent of hepatitis E, the latest discovered and the least explored of the major hepatic diseases in humans (Chandra et al. 2008; Panda et al. 2007). Among hitherto identified human hepatitis viruses, it is the only one detected in animals, therefore having a zoonotic potential (Pavio et al. 2010). The infection is transmitted enterically, mainly by the consumption of contaminated food or water (Yugo and Meng 2013), and ranges from an asymptomatic to a severe course, as described in pregnant women (Navaneethan et al. 2008) and immunocompromised patients (Kamar et al. 2011; Le Coutre et al. 2009). So far, four HEV genotypes (HEV-1 to HEV-4) infecting humans have been recognised within seven genotypes of species A from genus Orthohepevirus according to the classification proposed by Smith et al. (2014). HEV-1 and HEV-2 are human specific, while two genetically more diverse genotypes HEV-3 and HEV-4 are reported from both human and animal sources. Epidemiological patterns (Van der Poel 2014; Yugo and Meng 2013) reveal that genotypes 1 and 2 are hyperendemic in developing countries, while genotypes 3 and 4 have been reported mainly in industrialised regions and are responsible for sporadic human cases. Although genotypes HEV-3 and HEV-4 have been isolated from a broad range of animals (Meng 2010; Yugo and Meng 2013), only domestic swine, wild boar and deer should be considered as
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true reservoirs of the disease (Van der Poel 2014). Hitherto persistent circulation of the virus within a closed animal population has experimentally been confirmed only for domestic swine (Andraud et al. 2013; Bouwknegt et al. 2009) in which not only faecal-oral route, but also direct contact transmission has been demonstrated (Andraud et al. 2013; Kasorndorkbua et al. 2004). Also possibly aerosol transmission has been suggested recently (Bouwknegt et al. 2009). The HEV shedding pattern, high prevalence, in many countries exceeding 60 % (Van der Poel 2014), as well as the fact that swine do not develop clinical symptoms support the assumption that domestic swine may be the main HEV reservoir and that foodborne transmission of HEV may have an important role in HEV epidemiology. A few examples of hepatitis E have been associated with the consumption of raw or undercooked food products of swine, wild boar, deer or contaminated shellfish (Said et al. 2009; Tamada et al. 2004; Tei et al. 2003; Yazaki et al. 2003). Moreover, a statistically higher seroprevalence found in pig farmers and veterinarians in comparison to the general population (Krumbholz et al. 2012) suggests that a contact exposure to swine may also be a risk factor. Even though the infection in humans is generally self-limiting, the virus may be transmitted by blood transfusion and organ transplantation, as demonstrated recently (Kamar et al. 2012). HEV RNA sequences have been detected in sewage, surface and waste waters, and have been shown to have high genetic similarity with those found in indigenous human cases and in swine and wildlife from the same geographical regions (Van der Poel 2014). The current HEV detection is based on PCR methods. HEV single-stranded RNA genome of 7.2 kb consists of three open reading frames (Okamoto 2007; Tam et al. 1991), from which ORF1, encoding non-structural proteins, and ORF2, encoding a capsid protein, are mainly used for inferring the virus phylogeny. The recent serological study in Croatia confirmed antiHEV antibodies in 10 % of 439 tested patients with liver damage in which the absence of acute viral hepatitis A, B or C markers was confirmed (Dakovic Rode et al. 2013) demonstrating the possibility of zoonotic HEV transmission in the country. This hypothesis was further corroborated by the first HEV human case described in Croatia (Cˇivljak et al. 2013) proven to be autochthonous. To determine the potential sources of HEV in the country, we have performed a comprehensive epidemiological survey based on HEV RNA detection. HEV presence was tested in 2354 domestic and wild animals of a different age and sex, and representatives of ten animal species found in Croatia including molluscs (mussels and oysters) as indicators of a possible sea contamination. In order to determine the phylogenetic relationship of Croatian HEV strains with those already published,
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selected ORF1 and ORF2 amplicons were sequenced. Due to the complex RNA virus population structure (Ojosnegros et al. 2011) and in order to gain better epidemiological insight of HEV transmission routes, all amplicons were cloned prior to further analyses.
Methods Samples Samples of different symptomless animals bred in farms (domestic) or hunted during the hunting season (wild) were collected during 2009 and 2010 from different locations covering all Croatian counties (Fig. 1). In total, 2354 samples were analysed: 536 wild boars (Sus scrofa L.), 848 domestic pigs (Sus scrofa domestica), 32 cattle (Bos taurus), 40 roe deer (Capreolus capreolus), 280 red deer (Cervus elaphus), 12 muflons (Ovis musimon), 50 foxes (Vulpes vulpes), 10 martens (Martes martes), 8 ferrets (Mustela putorius), 252 mussels (Mytilus galloprovincialis) and 286 oysters (Ostrea edulis). Swine samples (all categories of domestic pigs and wild boars) were collected through a national classical swine fever monitoring programme, while other samples (cattle, roe and red deer, muflons, foxes, martens, ferrets and molluscs) were collected for diagnostics of different virus diseases routinely performed in Croatian Veterinary Institute. Digestive gland samples were collected from mussels and oysters, while blood, spleen and liver samples were collected from all other investigated animal species. Sera were separated from cellular elements by centrifuging coagulated blood (the blood clots were rimmed with a sterile glass stick to facilitate separation) for 15 min at 10009g. An amount of 140 ll of each serum sample was used for viral RNA purification using the QIAamp viral RNA extraction kit (Qiagen) according to the manufacturer’s instructions. Approximately, 0.1 g of spleen, liver and digestive gland samples were homogenised and diluted in 1 ml of phosphate-buffered saline (PBS; pH 7.4). The homogenised samples were then vortexed for 1 min and centrifuged for 15 min at 10009g. An aliquot (140 ll) of each sample supernatant was used for the viral RNA isolation by QIAamp viral RNA extraction kit (Qiagen) according to the manufacturer’s protocol. The RNA samples isolated from blood, spleen and liver of each sample, excluding molluscs, were pooled, and RNA was stored at -80 °C until needed. HEV RNA Detection The amplification of HEV ORF1 and ORF2 fragments was adapted from previously published methodology (Huang
Food Environ Virol Fig. 1 Geographical origin of the examined animal samples originated from different Croatian counties. Negative samples are marked with empty and positive samples with filled shapes
et al. 2002; Van der Poel et al. 2001). Six microlitres of RNA extracts was used for reverse transcription by SuperScript III reverse transcriptase using random hexamers (Invitrogen). Two pairs of HEV primers were used for direct and nested PCR amplification of 287-nucleotidelong product of ORF1 (ORF1-s1/ORF1-a1—direct and ORF1-s2/ORF1-a2—nested) (Wang et al. 1999) and 348-nucleotide-long product of ORF2 region (ORF2-3156/ ORF2-3157—direct and ORF2-3158/ORF2-3159—nested) (Huang et al. 2002). The reaction mixtures of 50 ll containing 6 ll of cDNA were prepared following the manufacturer’s instructions (PlatinumÒ Taq DNA polymerase, Invitrogen). The direct and nested PCR parameters for the optimised amplification assay were mutually identical. The amplification procedure for ORF1 region was as follows: denaturation at 94 °C for 3 min; incubation at 94 °C for 45 s, 55 °C for 45 s, and 72 °C for 45 s (35 cycles); and incubation at 72 °C for 20 min. ORF2 region amplification procedure was denaturation at 94 °C for 3 min; incubation at 94 °C for 3 min, 50 °C for 1 min, and 72 °C for 1 min (30 cycles); and incubation at 72 °C for 20 min. PCR products were
separated by agarose gel electrophoresis in 1.5 % agarose gel stained with ethidium bromide and visualised by UV transillumination. All standard precautions were followed to prevent PCR contamination. For the verification of the results, no template, as well as positive control, was included in each amplification experiment. As a positive control, HEV strain that had previously been characterised was used. HEV Genotyping To determine the phylogenetic clustering of Croatian HEV strains, eleven positive swine samples, randomly selected representatives of different counties, were selected for further genotyping (Figs. 3, 4). Different genomic variants present in RNA virus populations were separated by TA cloning of nested PCR amplicons into the pTZ57R/T vector (Fermentas) as described by the manufacturer. The transformation of competent Escherichia coli XL-1 Blue cells was done using commercial InsTAcloneTM PCR cloning kit (Fermentas). Transformed cell colonies were selected by a-complementation, and the
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presence of the insert was confirmed by PCR using the same primer pairs and nested PCR protocols as previously described. Three clones per sample transformed with ORF2 as well as three clones per sample transformed with ORF1 amplicons were chosen randomly. Plasmids were purified using a PureLinkTM Quick Plasmid Miniprep Kit (Invitrogen), and inserts were sequenced in both directions (Macrogen) using a pair of M13-pUC universal primers. To determine the phylogenetic clustering of the obtained sequences, the prototype ORF1 and ORF2 sequences, representing all proposed Orthohepevirus A genotypes (Smith et al. 2014) were retrieved from GenBank. Also, similar sequences obtained using BLAST algorithm were included in the analysis. Sequences were aligned using ClustalX, version 2.0 (Thompson et al. 1997) and analysed using MEGA 5 (Tamura et al. 2011). Phylogenetic trees were generated using the neighbour-joining method applying Tamura-Nei evolutionary model. The tree topology was evaluated by bootstrap analysis based on 1000 repetitions. Statistical Analysis For the comparison of categorical variables between groups, v2 test was used. A p value of B0.05 was considered statistically significant.
Results Virus Detection Both HEV ORF1 and ORF2 amplicons were detected from pooled samples in 24.5 % of tested domestic pigs and in 12.3 % of tested wild boars. All positive animals originated from the north and north-east Croatian regions including 7 (Virovitica Podravina, Osijek Baranja, Vukovar Srijem, Bjelovar Bilogora, Brod Posavina, Sisak Moslavina and Koprivnica Krizˇevci) and 4 (Virovitica Podravina, Vukovar Srijem, Brod Posavina, Sisak Moslavina) counties, respectively (Fig. 1). The HEV prevalence detected in the counties where positive samples were detected ranged from 13.3 to 57.9 % for domestic pigs and from 20 to 31.8 % for tested wild boars (Table 1). HEV was found in all age categories, regardless of sex or breeding conditions. However, most of the positive samples (Fig. 2) were found within domestic pigs up to 6 months of age and wild boars up to 1 year of age. Even though the difference of HEV incidence among age groups of domestic pigs and wild boars was highly significant, the geographical distribution was unrelated to the incidence of positive samples from both groups (p [ 0.05 and p = 0.65, respectively). HEV
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RNA was not detected in other animal and molluscs samples. In each amplification experiment, clear amplification signal was detected when using positive control and no amplification when using no-template control. Sequence Analysis The sequence analysis of ORF1 and ORF2 clones confirmed the coexistence of different genomic variants in all tested samples but one (CRO OB F). Obtained sequences (i.e., CRO SM W b) were marked by country of origin (CRO), twoletter acronyms representing county, mark indicating the type of habitat (F-domestic farm animals, D-domestic backyard animals, W-wild boars) and small letter (a–c) for a clone number. All sequences displaying nucleotide differences obtained in this work were submitted to GenBank excluding primer regions. Accession numbers are as follows: KJ850446–KJ850476, KF366506–KF366510, KF366514– KF366518 and KF366520–KF366524. The phylogenetic relationship analyses based on both ORF1 and ORF2 amplicons confirmed that all sequences obtained from domestic pigs and wild boars clustered into genotype 3 (Figs. 3, 4) of Orthohepevirus A according to the classification proposed by Smith et al. (2014). Based on both ORF1 and ORF2 phylogenies supported by the high bootstrap values, the sequences of the majority of Croatian strains clustered into one of two subgroups: cluster CRO A (CRO VS W, CRO VS D, CRO OB F, CRO BB D, CRO SM W, CRO SM D) and cluster CRO B (CRO BP W, CRO BP D). Also, based on both ORF1 and ORF2 phylogenies, in sample CRO VP F, sequences clustering into both subgroups (CRO A and CRO B) were detected. The two inconsistencies were found in samples CRO VS F and CRO KK D. In samples CRO VS F, based on ORF1 phylogeny, the sequences clustered into subgroup CRO A, while based on ORF2 phylogeny, sequences clustering into both subgroups were detected. However, in sample CRO KK D based on ORF1 phylogeny, the variants clustered into subgroup CRO B, while based on ORF2 phylogeny, they clustered into subgroup CRO A. The comparison of Croatian ORF1 sequences (Fig. 3) with reference database sequences showed the highest genetic similarities with Hungarian wild boar (97.5 %; EU718647), German human (97.5 %; FN994999), Serbian swine (93.1 %; HM483380) and USA human (91.8 %; JN837481) strains. Croatian ORF2 sequences (Fig. 4) displayed the highest genetic similarity with Japanese human (95.9 %; AB089824), USA swine (94.4 %; AF466682) and Hungarian human (94.4 %, EF530669) HEV strains. By comparing sequences of different Croatian strains mutually, 100 % nucleotide identity was found in wild and domestic animals (grown on farms and backyards) from the same county (Vukovar Srijem) as well as from wild and
Food Environ Virol Table 1 The list of domestic and wild animals from different Croatian counties tested for the presence of HEV RNA County Virovitica Podravina (VP) Osijek Baranja (OB)
Vukovar Srijem (VS)
Bjelovar Bilogora (BB)
Brod Posavina (BP)
Sisak Moslavina (SM)
Koprivnica Krizˇevci (KK)
Med¯imurje (M)
Varazˇdin (V) Krapina Zagorje (KZ)
Zagreb (ZG)
Pozˇega Slavonija (PS)
Karlovac (K)
Primorje Gorski kotar (PG)
Species (habitat)
Number of positive results/tested animals
Percentage of positive samples (%)
Pig (farm)
38/115
33.0
Wild boar
9/30
30.0
Pig (farm)
70/336
20.8
Wild boar
0/84
0
Cattle
0/10
0
Roe deer, Red deer
0/7, 0/40
0
Pig (farm)
13/50
26.0
Pig (backyard)
20/58
34.5
Wild boar
6/30
20.0
Roe deer, Red deer, Muflon
0/10, 0/50, 0/4
0
Pig (backyard)
2/15
13.3
Cattle
0/10
0
Red deer, Muflon
0/80, 0/3
0
Pig (backyard)
7/18
38.8
Wild boar
21/66
31.8
Roe deer, Red deer
0/10, 0/70
0
Pig (backyard)
55/95
57.9
Wild boar
30/95
31.6
Fox
0/9
0
Pig (backyard)
3/15
20.0
Wild boar
0/33
0
Cattle
0/10
0
Pig (backyard)
0/10
0
Wild boar
0/21
0
Fox
0/10
0
Pig (backyard)
0/10
0
Fox, Ferret, Marten
0/5, 0/8, 0/5
0
Pig (backyard)
0/14
0
Wild boar
0/19
0
Roe deer
0/5
0
Pig (farm)
0/55
0
Pig (backyard)
0/10
0
Wild boar
0/20
0
Fox, Marten, Roe deer
0/10, 0/5, 0/8
0
Pig (backyard)
0/18
0
Wild boar
0/42
0
Fox
0/6
0
Pig (backyard)
0/15
0
Wild boar
0/30
0
Fox
0/10
0
Pig (backyard)
0/8
0
Wild boar
0/23
0 0
Cattle
0/12
Istria (I)
Red deer, Muflon, Mussel, Oyster
0/40, 0/5, 0/51, 0/97
0
Lika Senj (LS)
Wild boar
0/43
0
Zadar (ZD) Sˇibenik Knin (SˇK)
Mussel, Oyster
0/48, 0/96
0
Mussel
0/67
0
Split Dalmacija (SD)
Pig (backyard)
0/6
0
Dubrovnik Neretva (DN)
Mussel, Oyster
0/86, 0/93
0
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% of positive samples
Food Environ Virol
30,00 25,00 20,00 15,00 10,00 5,00 0,00
age groups domesc pigs
wild boars
Fig. 2 The prevalence of HEV infection in wild boars and domestic pigs detected by nested RT-PCR and categorised in different age groups. Wild boars category and the number of tested samples: shoats (\1 year, 187), yearlings (1–2 years, 176), adults ([2 years, 173). Domestic pigs category and the number of tested samples: sucklings
(1–4 weeks, 119), weanlings (5–10 weeks, 110), fatlings ([10 weeks, 256) and domestic pigs older than 6 months (boars 108; gilts 121; sows 139). For comparison of categorical variables between groups, v2 test was used
domestic animals (grown in backyards) from the same county (Brod Posavina and Sisak Moslavina; Figs. 3, 4— diamond markings). Moreover, 100 % nucleotide identity was found in all three categories of animals originating from different counties: (i) in farm animals from counties Virovitica Podravina, Vukovar Srijem and Osijek Baranja; (ii) in backyard animals from counties Vukovar Srijem, Bjelovar Bilogora and Koprivnica Krizˇevci, as well as from counties Virovitica Podravina and Koprivnica Krizˇevci; and (iii) in wild boars from counties Sisak Moslavina and Vukovar Srijem.
zoonotic HEV genotypes among wild animals. As the HEV monitoring programme has not been established in the country yet, samples used in this study were collected through a national classical swine fever monitoring programme or other programmes for monitoring common animal viruses. According to our RT-PCR results, HEV RNA was detected in all age groups of domestic pigs and wild boars sampled in Croatia. The correlation of both positive and negative results obtained using ORF1 and ORF2 primer sets was 100 % for all tested samples. The high sensitivity of the RT-PCR assay used was previously confirmed by Huang et al. (2002) and Wang et al. (1999) testing the sensitivity of the assay using the same ORF2 and ORF1 primer sets, respectively. Interestingly, HEV RNA was not confirmed in any other domestic or wild animal included in this study. Part of the explanations could be that the primer sets used are not specific for HEV genotypes belonging to more distantly related HEV species -B, -C or -D, (Smith et al. 2014), amongst which HEV variants from wild animals, such as ferret and mink, were lately identified. Nevertheless, the zoonotic potential of these strains has not been reported, and therefore their detection was not our priority. On the other hand, our sequence analysis indicates that at least one of the primer sets used would amplify HEV variants from all genotypes described within genus Orthohepevirus A, four of which have zoonotic potential. The average HEV prevalence in swine samples appeared to be quite high; 24.5 % of domestic swine and 12.3 % of wild boars proved to be HEV RNA positive with the
Discussion In the last decade, a few sporadic cases of foodborne HEV infection through the consumption of uncooked contaminated animal products and shellfish (Koizumi et al. 2004; Said et al. 2009; Tamada et al. 2004; Tei et al. 2003), but also epidemic outbreaks due to contaminated water or water supplies (Ceylan et al. 2003; El-Esnawy 2000; Rodriguez-Lazaro et al. 2012), have been reported. However, the cause of infection in many autochthonous sporadic cases reported in low or non-endemic countries mostly remain unidentified. In our study, samples from different wild and domestic animals, as well as molluscs, all potential HEV reservoirs, used for food were examined for the presence of HEV RNA. Furthermore, muflons, foxes, martens and ferrets were included in the analysis with the aim to explore their possible role in the spread of the
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Food Environ Virol CRO VS W c
Fig. 3 Neighbour-joining phylogenetic tree obtained by the analysis of HEV ORF1 sequences (242 nt) of genomic variants from Croatian wild boar and domestic pig HEV samples. Genotype reference sequences were adopted from Smith et al. (2014). As an outgroup, HEV B prototype sequence (Smith et al. 2014) was used. Identical sequences found in the different animal groups from the same county were marked (diamond symbol). Bootstrap values are presented next to tree nodes. The bar represents 0.005 nucleotide substitution per site
CRO VS D a CRO OB F a 14
CRO VS D b CRO VS W a
99
CRO VP F a CRO BB D
22
CRO OB F b 6
HM483380 CRO SM D a JN837481
17
cluster CRO A
AF082843
39
CRO SM D b
71
3
44 CRO SM W b 99
CRO VS W b
77
CRO SM W a CRO SM D c
16
39
HM483383 EU718646
23 26
46
AB108666 EU718647 38
AY115488
GENOTYPE 3
AB301710
80
AB114179
57
AF336013
40 56
FJ705359
95
AB290312
26
AF455784 AF195064
48
HM052802
19
JF431046 EU718650
22
JN415693 68
AF110389 95
FN994999
48
64 CRO VS F b
CRO KK D a
93
19
CRO BP W CRO VS F a
91
CRO BP D a
cluster CRO B
61 CRO KK D c
CRO VP F b
28
6 CRO BP D b 31
CRO KK D b
61 76
AB573435
GENOTYPE 5
AB602441
GENOTYPE 6
KJ496143
GENOTYPE 7
AJ272108
GENOTYPE 4
M74506 35
GENOTYPE 2
M73218
GENOTYPE 1
AY535004
HEV B (outgroup)
0.05
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Food Environ Virol CRO VP F b
Fig. 4 Neighbour-joining phylogenetic tree obtained by the analysis of HEV ORF2 sequences (301 nt) of genomic variants from Croatian wild boar and domestic pig HEV samples. Genotype reference sequences were adopted from Smith et al. (2014). As an outgroup, HEV B prototype sequence (Smith et al. 2014) was used. Identical sequences found in the different animal groups from the same county were marked (diamond symbol). Bootstrap values are presented next to tree nodes. The bar represents 0.05 nucleotide substitution per site
CRO KK D CRO BB D 64
CRO OB F CRO VS D a
100
CRO VS F b CRO VS W
47
cluster CRO A
CRO VS D b CRO SM W a CRO SM D a CRO SM W b
100
41
46 CRO SM D b 44
CRO SM D c
AB089824
74
AB094270
15
AF466682
56
25
AF082843 AB290312
41
GENOTYPE 3
FJ705359
20
AF336290
90 45
AB301710 AF336292 AF455784
49
AB094231
59
EU684329
84
EF530669
68
CRO VS F c
92 9
CRO BP W a
100
CRO BP D a
68 20 66 24
CRO BP W b CRO VP F a
cluster CRO B
CRO VS F a
12 CRO BP D c 16 CRO VP F c
0
21 CRO BP D b
AB602441
GENOTYPE 6
KJ496143
13 17
AB573435 AJ272108
71
GENOTYPE 5 GENOTYPE 4
M74506
33
GENOTYPE 7
M73218 AY535004
GENOTYPE 2 GENOTYPE 1 HEV B (outgroup)
0.05
highest prevalence (Table 1) reported in two neighbouring counties Sisak Moslavina and Brod Posavina reaching 57.9 % in domestic swine and 31.8 % in wild boar populations. Since the sample set available for testing was not the representative of the distribution of domestic pigs and wild boar in different Croatian counties, in some cases, the results might have been affected by a small sample size. A previous study in Croatia (Lipej et al. 2013), even in a small amount of tested samples, confirmed an extremely high seroprevalence of HEV-specific antibodies in the domestic swine population (91.7 %). Therefore, a high
123
prevalence of the viral RNA was expected, even though the viral RNA prevalence reported by Lipej and collaborators (2013) was relatively low (13.3 % for serum and 8.1 % for bile samples, respectively). A high HEV prevalence is not uncommon in many European countries (Berto et al. 2012; Ferna´ndez-Barredo et al. 2007; Martelli et al. 2008; Reuter et al. 2009; Rutjes et al. 2007; Schielke et al. 2009), among which the prevalence reported in Hungary (Reuter et al. 2009) and Spain (Ferna´ndez-Barredo et al. 2007) were the most similar to the one reported in domestic swine in Croatia. However, the HEV prevalence reported in wild
Food Environ Virol
boars was the most similar to those reported in Germany (Schielke et al. 2009) and Hungary (Reuter et al. 2009). All positive samples were geographically located in the north and north-east part of the country, with positive domestic pigs found in seven counties and wild boars found in four of them (Fig. 1). In wild boars, the incidence distribution found by age is consistent with the dynamics described for HEV infection in Germany (Schielke et al. 2009). The reason why we have not detected the virus in the yearlings category (Fig. 2) might be relatively small number of tested animals from that age group. As described by Pavio et al. (2010), the peak of viremia in domestic swine was usually observed at 15 weeks of age corresponding to our fatlings category (Fig. 2). However, our data show the highest prevalence of HEV infection in the age class of 1–4 weeks (Fig. 2 sucklings). HEV infections in very young piglets have previously been reported (de Deus et al. 2008), but the explanation is still unclear. Possible reasons could be the horizontal sow-to-piglet transmission, which in our case is corroborated by the high HEV prevalence in sows category, or transplacental transmission as which so far has not been confirmed experimentally in swine (de Deus et al. 2008). Since positive animals have been found amongst all age groups of animals used for human consumption, this finding raises further public health concerns. The sequence analysis of cloned ORF1 and ORF2 amplicons, even in a small number of clones tested, revealed the complex HEV populations consisting of different genomic variants in all but one analysed sample, which is in accordance with quasispecies nature of RNA virus populations (Domingo et al. 2012). The phylogeny of both ORF1 and ORF2 regions (Figs. 3, 4) confirmed the clustering of Croatian strains into genotype 3, that is, excluding a few sporadic findings of genotype 4, a common HEV genotype found in Europe (Riveiro-Barciela et al. 2013). Although the latest publications (Olivera-Filho et al. 2013; Smith et al. 2014) do not support firm subgenotype classification, based on the one published by Lu et al. (2006) and Steyer et al. (2011), Croatian cluster CRO A would be classified as subgenotype 3a, whilst Croatian cluster CRO B would be classified as subgenotype 3e. The additional ORF2 sequence variants belonging to different clusters obtained in sample CRO VS F could simply be explained by a small number of clones analysed in this study. However, the explanation of the complete inconsistency found in the clustering of ORF1 and ORF2 sequence variants of sample CRO KK D could be the recombination, which, due to the complex within-host virus populations of RNA viruses, is not a rare event. Interestingly, in three Croatian counties (Virovitica Podravina, Vukovar Srijem and Koprivnica Krizˇevci), animals co-infected with virus variants from two subgroups were found. However, the pattern of
clustering into subgroups could not be linked with geographical distribution and was not species specific since both subgenotypes were detected in domestic swine, from both farms and backyards, as well as from wild boars. Even so, the results of mutual comparison of Croatian HEV sequences exposed a few possible HEV transmission events within the country, while identical sequence variants found in farm pigs from three neighbouring counties (Osijek Baranja, Vukovar Srijem and Virovitica Podravina) may be related to the introduction of breeding animals from the same, previously infected source; the finding of identical sequence variants in farm and backyard swine, as well as in wild boars in the same county (Vukovar Srijem), is most probably the result of environmental transmission. This hypothesis seems to be applicable in two more counties (Brod Posavina and Sisak Moslavina) where identical sequence variants were found in domestic swine grown in backyards and in wild boars. Moreover, identical sequence variants were found in tested animals originating from different counties. They were found in backyard pigs from neighbouring counties (Bjelovar Bilogora and Koprivnica Krizˇevci; Virovitica Podravina and Koprivnica Krizˇevci), but also in wild boars from non-neighbouring counties (Sisak Moslavina and Vukovar Srijem). The most likely scenario is that the virus was transmitted from a farm to domestic pigs and further on to wild boars due to strict bio-security measures implemented on farms that would prevent a vice versa course of infection. The accurate source of infection in domestic pigs is difficult to define due to an intensive import from different European countries in the past decades. However, the comparison of Croatian HEV sequences with those reported in GenBank indicates the highest sequence similarity of Croatian strains with those from Serbia and Hungary, which are bordering countries. To understand this complex epidemiological scenario, more detailed molecular epidemiology studies encompassing a higher number of samples from each geographical location and a higher number of clones analysed per sample would be necessary. The comparison of Croatian HEV sequences with the reference sequences from GenBank revealed a surprising finding that a high sequence similarity was reported among Croatian swine sequences and those from human HEV strains originating from Germany, Japan, Hungary and USA. This strongly suggests the possible zoonotic transmission of Croatian HEV variants described here, especially after the first autochthonous human HEV in Croatia was diagnosed (Cˇivljak et al. 2013). Moreover, a recent serological study (Dakovic Rode et al. 2013) confirmed anti-HEV antibodies in even 10 % of 439 tested patients with liver damage and the absence of acute viral hepatitis A, B or C markers in Croatia. To complete the general epidemiological picture of HEV transmission in Croatia,
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Food Environ Virol
thorough analyses of surface waters from the vicinity of large industrial and small backyard pig holdings, positioned mainly in the continental part of the country, would provide reliable information regarding the HEV presence in the environment and probable routes of environmental virus transmission. Moreover, further studies on the role of domestic pigs and wild animals in HEV transmission chain are necessary to develop measures for prevention of zoonotic and environmental transmission to humans and other animal species. The fact that HEV RNA was not detected in other domestic or wild animals cannot, however, exclude them to be involved in the virus shedding, since so far only pigs have been recognised as truth HEV reservoirs. Nevertheless, the presence of HEV within the wild boar population may allow further spread of disease to other wild animals and distant locations increasing the risk of possible human infection. In conclusion, our results confirm that the genotype 3 of HEV RNA was detected in all major swine growing regions in Croatia indicating that domestic pigs and wild boars may be important reservoirs of HEV in the country presenting a significant risk for HEV infection in humans. Acknowledgments We thank Ivana Prajdic´ and Marin Jezˇic´ for their technical assistance. This study was supported by the Croatian Ministry of Science, Education, and Sports (Project No. 048-0481186-1183).
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