Hepatitis E virus infection in 6-month-old pigs in Taiwan

Hepatitis E virus (HEV) is the causative agent of acute hepatitis E. Genotype 3 (G3) and 4 (G4) HEV have recently been identified in and isolated from swine as the main HEV genotypes worldwide. However, there is limited information on HEV infection status among pigs in Taiwan, especially pigs in the stage before transportation to the slaughterhouse. To determine the frequency of HEV infection among pigs in Taiwan, we detected and quantified HEV RNA contained in 295 fecal specimens collected from 6-month-old pigs bred in 30 pig farms located in 8 counties. We found that 25.1% (74/295) of the fecal specimens were positive for HEV RNA by a quantitative real-time reverse transcription-polymerase chain reaction, and the copy number ranged from 2.3 × 103 to 2.08 × 107 copies/g. Amplification of a 338 bp sequence in ORF2 was achieved in 16 of 74 HEV RNA-positive samples, and their nucleotide sequences were determined. Two HEV sequences appeared to belong to subtype 3a of G3 and the remaining 14 HEV sequences belonged to subtype 4b of G4 (G4b). The entire genome sequence of two G4b HEVs was obtained by next-generation sequence analyses, and the phylogenetic analyses indicated that unique G4b HEVs were circulating in pig farms in Taiwan. In the present study, we found that both G3 and G4 HEVs were circulating in Taiwanese pig farms and G4b was the predominant subtype. In addition, the relatively high detection frequency of HEV RNA in the 6-month-old pigs indicated that Taiwanese pigs just before transportation to the slaughterhouse are at risk of carrying HEVs, and thus thorough cooking or heating of pork meat or organs is needed before consumption in Taiwan and possibly in other countries as well.


Scientific Reports
| (2020) 10:16869 | https://doi.org/10.1038/s41598-020-74034-8 www.nature.com/scientificreports/ Swine are thought to be the main reservoir of G3 and G4 HEV 12 . Because HEV-infected pigs excrete large quantities of HEV into the feces, zoonotic transmission of HEV could occur through direct contact with pigs. In fact, the antibody positive rate against HEV was found to be 1.51 times higher in veterinarians handling pigs than in normal blood donors 13 , and was also higher among swine farmers than the general population 14 . Because HEV replicates in the liver and the transient viremia is associated with the dissemination of HEV into muscle and other tissues, consumption of uncooked or undercooked liver, meat or related products from HEV-infected pigs might confer a risk of HEV transmission in humans 15 . Therefore, we investigated the current infection status of HEV in the pigs just before transportation to the slaughterhouse. Our findings should be useful for the risk assessment and management of viral hepatitis due to HEV.

Materials and methods
Sample collection. A total of 295 swine fecal specimens were collected from 30 commercial farms (F1 to F30) in Taiwan from January 12 to December 13, 2015 ( Table 1). The swine farms were located in 8 counties: Pingtung (F1-4, F6, F9-11, F14, F17, and F29), Changhua (F13, F15, F16, F18, F20, F27, F28, and F30), Miaoli (F25, F26), Yunlin (F12, F23, and F24), Taoyuan (F21 and F22), Taitung (F19), Taichung (F5), and Kaohsiung (F7 and F8). Ten samples were collected from each farm, except 2 farms where 7 (F8) and 8 (F23) samples were collected ( Fig. 1 and Table 1). All of the pigs were 6 months old and therefore in the terminal fattening stage before shipping. Three grams of fecal specimens were directly collected from individual swine and diluted with 10 mM phosphate-buffered saline (PBS) to prepare a 10% (w/v) suspension. The suspension was shaken at 4 °C for 1 h, clarified by centrifugation at 10,000 × g for 30 min, passed through a 0.45 µm membrane filter (Millipore, Bedford, MA), and stored at − 80 °C until use 16 . The experiments were reviewed and approved by the Taiwan Centers for Disease Control (CDC) ethics committee and all of the animal experiments were carried out according to the Guides for Animal Experiments Performed at Taiwan CDC.   www.nature.com/scientificreports/ used as the standard to quantitate the copy numbers. G3 HEV was originally isolated from the fecal specimen of a pig and cultured in a human hepatocarcinoma cell line, PLC/PRF/5. cDNA was produced from isolated virus RNA, and the full genome (AB740232) was cloned into pUC19 under the T7 promoter 18 . The capped G3 HEV RNA was synthesized using an mMESSAGE mMACHINE T7 kit (Ambion, Austin, TX) and the copy number was calculated based on the RNA concentration and molecular weight. Amplification data were collected and analyzed with Sequence Detector software ver. 1.3 (Applied Biosystems). The detection limit was 10 3 copies/ml.

RT-PCR for amplification of the HEV genome.
Reverse transcription was performed with a highcapacity cDNA reverse transcription kit (ABI Applied Biosystems, Foster City, CA) at 25 °C for 10 min, 37 °C for 120 min and 85 °C for 5 min in a 20 µl reaction mixture containing 1 µl of reverse transcriptase, 2 µl of the random primer, 1 µl of RNase inhibitor, 2 µl of 10 × RT buffer, 0.8 µl of 10 mM deoxynucleoside triphosphates, 8 µl of RNA, and 5.2 µl of distilled water 19 . A nested polymerase chain reaction (PCR) was performed to amplify a portion of the open reading frame 2 (ORF2) genome. The first PCR was carried out with an external forward primer, HEV-F1 (5′-TAYCGHAAY CAA GGHTGGCG-3′), and an external reverse primer, HEV-R2 (5′-TGY TGG TTR TCR TAR TCC TG-3′). The amplification was carried out for 35 cycles (95 °C for 30 s, 55 °C for 45 s, and 72 °C for 90 s) after a denaturation at 95 °C for 60 s and followed by a final extension at 72 °C for 7 min. Two microliters of the first PCR product were used for the nested PCR with an internal forward primer, HEV-F2 (5′-GGBGTBGCNGAG GAG GAGGC-3′), and an internal reverse primer, HEV-R1 (5′-CGA CGA AAT YAA TTC TGT CG-3′), under the same amplification conditions as used for the first PCR. The monkey fecal samples collected before-and post-G1 HEV infection were used as the negative and positive control for RT-PCR, respectively. The detection limit was determined to be 10 4 copies/ml by real-time RT-qPCR. The nested PCR products with 378 bp nucleotides were separated by electrophoresis on 2% agarose gels 20 .
HEV genome sequencing. The PCR products were purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany), and the nucleotide sequencing was carried out with primers HEV-F2 and HEV-R1 using an ABI 3130 Genetic Analyzer Automated Sequencer (Applied Biosystems, Foster City, CA) and a BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems) according to the manufacturer's instructions. Sequence analysis was performed using the Genetyx ver.11.0.4 software program (Genetyx Corp., Tokyo).

Next-generation sequence analysis (NGS).
The entire genome sequences were determined by NGS as described previously 21 . Briefly, the viral RNA was extracted from the 10% fecal specimens, and a 200 bp fragment library was constructed with a NEBNext Ultra RNA Library Prep Kit for Illumina version 2.0 (New England Biolabs, Ipswich, MA) according to the manufacturer's instructions. Library purification was done using Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA). A 151-cycle paired-end read sequencing run was carried out on a MiSeq desktop sequencer (Illumina, San Diego, CA) using an MiSeq Reagent Kit version 2 (300 cycles). Sequence data were analyzed using CLC Genomics Workbench Software version 7.5.1 (CLC Bio, Aarhus, Denmark).

Phylogenetic analyses.
Phylogenetic trees with 1,000 bootstrap replicates were generated by the neighbor-joining method based on the partial ORF2 sequence (338 bp) or entire HEV genome. Bootstrap values of 95 or higher were considered statistically significant for the grouping 22 . The nucleotide sequence alignment was performed using Clustal X 1.81. The genetic distance was calculated by Kimura's two-parameter model 23 .

Characterization of HEV in pig fecal specimens.
To determine the frequency of HEV infection among pig populations in Taiwanese farms, we detected and quantified HEV RNA in 295 fecal specimens collected from 6-month-old pigs bred in 30 pig farms in 8 counties in Taiwan (Fig. 1). HEV RNA was detected in 23 out of 30 farms: 0% (0/10) in Taitung (Table 1). We found that 25.1% (74 of 295) of the fecal specimens were positive for HEV RNA by real-time RT-qPCR, and the copy number ranged from 2.3 × 10 3 to 2.1 × 10 7 copies/g.
Amplification of a 378 bp sequence in ORF2 was achieved in 16 of 74 HEV RNA-positive samples, and their nucleotide sequences were determined (GenBank accession nos. LC436678-LC436692, LC436449, and LC436450). Phylogenetic analyses indicated that 14 sequences belonged to G4, subtype 4b (G4b) (Fig. 2); these were 2 sequences from farm F3 in Pingtung; 1 from farm F7 and 1 from farm F8 in Kaohsiung; 3 from farm F15 in Changhua; 6 from farm F22 in Taoyuan; and 1 from farm F23 in Yunlin County (Fig. 1, Table 1). The interfarm nucleotide sequence identities between the 6 farms were 89.5% to 99.7%, while the intra-farm nucleotide sequence identities were as high as 99.4% to 100%. These 14 G4b HEVs shared 90.4% to 94.0% nucleotide sequence identity with those detected in the serum from hepatitis patients (AF296161 and AF117277) and 90.7% to 93.5% nucleotide sequence identity with those detected in the serum from swine (EU497922 and AF117280) in Taiwan, respectively. The remaining 2 HEV sequences collected from farm F25 in Miaoli County were identical and belonged to G3, subtype 3a (G3a), sharing 90.5% to 92.9% nucleotide sequence identity with the G3a HEV sequences detected in Taiwan, Japan and the USA (Fig. 2). These results demonstrated that several genetically different HEVs were circulating in the pig farms in Taiwan. . We found 4 nucleotide differences between them (C1091T, C4355T, C6355T and T6715C), and the nucleotide sequence identity was 99.9%, although the amino acid sequences of ORF1, ORF2 and ORF3 were identical. Phylogenetic analyses based on the entire genome demonstrated that these 2 HEVs, F22-1372 and F22-1380, belonged to G4b (Fig. 3). When we compared these 2 Taiwanese HEVs with 7 known G4b strains isolated in www.nature.com/scientificreports/ Japan, China and Cambodia, they were further separated into 3 clusters: G4b-1, which included 2 strains isolated from patients in Japan; G4b-2, which included 4 strains detected in pigs and rhesus monkeys in China and human patients in Cambodia; and G4b-3, which included 2 Taiwanese HEVs. The 2 Taiwanese HEVs analyzed in the present study shared 86.9-86.9% and 87.5-87.9% nucleotide sequence identities with G4b-1 and G4b-2, respectively, and formed a separate cluster, suggesting that G4b HEV is genetically diverse, and unique G4b HEVs were circulating in pig farms in Taiwan.

Discussion
Swine is a major reservoir of G3 and G4 HEV, and consumption of pig-derived foods is a potential source of zoonotic HEV infection 24−28 . Generally, HEV infection occurs after the weaning stage, and HEV RNA is detected mainly in serum samples in 3-to 4-month-old pigs in the farms 29,30 . The anti-HEV IgG-positive rates were shown to be as high as 90%, and no HEV RNA was detected in the serum samples in 6-month-old pigs 30 . However, Yazaki et al. tested packages of raw pig liver sold in grocery stores as food in Hokkaido, Japan, and found that 7 of 363 (1.9%) packages were positive for HEV RNA 31 . In the United Kingdom, the prevalence of the antibodies to HEV was 92.8% in pigs at the time of slaughter, and HEV RNA was detected in 15% of cecal contents and 3% of plasma samples in these pigs 32 . Moreover, the entire genome of G3 HEV was detected in the liver of a fattening pig in Switzerland 33 . In addition, the HEV RNA genome was detected in pork products such as meats, liver sausages and liver paté in Switzerland, Canada and France 26,34,35 . These results suggested that slaughter pigs and pork products are at risk of carrying HEV to humans. Further studies to explore the status of HEV infection www.nature.com/scientificreports/ may help to elucidate the potential risk of type E hepatitis deriving from the pigs before transportation to the slaughterhouse.
Because the rearing period of pigs is 6 months, we collected the fecal specimens from 6-month-old pigs in 30 farms in Taiwan, and found that 23 out of 30 farms were exposed to HEV and 25.1% of the pigs were positive for HEV RNA. This unexpectedly high prevalence of HEV RNA in the 6-month-old pigs obtained in the present study confirmed that the pigs before transportation to the slaughterhouse have a high risk for the spread of HEV infection. Although we exclusively examined HEV RNA by using fecal specimens, other tissues, such as meats, intestine or liver, must also be examined for HEV RNA after transportation to the slaughterhouse in order to evaluate the contamination of HEV.
Although a total of 74 fecal samples were positive for HEV RNA by real-time RT-qPCR, the amount of HEV RNA was lower than 10 4 copies/g in most of the samples. These results indicated that the copy numbers of the HEV genomes in the feces of 6-month-old pigs were low. However, we detected copy numbers as high as over 10 7 copies/g of RNA in two pigs (F22-1372 and F22-1380) in Taoyuan County, suggesting the possibility of super spreaders even in the final fattening stage of the pigs. In addition, the entire HEV genome was obtained from the feces of those two pigs. Phylogenetic trees were constructed based on both the partial ORF2 sequence (338 bp) and the entire HEV genome, and they showed that F22-1372 and F22-1380 were segregated into the subtype G4b. Therefore, there is no discrepancy in their constellation between the trees.
G3 and G4 HEV have been detected in hepatitis patients and pigs in Taiwan 36−39 , but the genetic information was limited, particularly for the entire genome of HEV. Our phylogenetic analyses based on the partial ORF2 sequences of the 16 HEVs revealed that both G3 and G4 HEV were circulating in pig farms in Taiwan. The G3a genome was detected in only 1 pig farm, while G4b was detected in 6 farms, suggesting that G4 HEV is more prevalent than G3 HEV in the Taiwanese pig farms.
In summary, our findings demonstrated the high prevalence of HEV in 6-month-old pigs in Taiwan, and suggested that pigs before transportation to the slaughterhouse are at a high risk of carrying HEV to humans. Since HEV could be inactivated by heating 40,41 , thorough cooking or heating is highly recommended before consumption of pork, pork liver, pork intestine or other related products to reduce the zoonotic infection due to HEV.