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J Vet Med Sci. 2024 May; 86(5): 524–528.
Published online 2024 Mar 29. doi:10.1292/jvms.23-0478
PMCID: PMC11144545
PMID: 38556348
Wenjing ZHANG,1 Yen Hai DOAN,2 and Tian-Cheng LI1,*
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Abstract
To conduct an epidemiological study of hepatitis E virus (HEV) in Japanese wild boars, we collected 179 serum and 162 fecal specimens from wild boars in eight Japanese prefectures; 39 ofthe serum samples (21.8%) were positive for anti-HEV IgG antibodies. RT-qPCR revealed HEV RNA in 11 serum samples (6.1%) and 5 fecal samples (3.1%). We obtained 412 bp of the viral genomesequences of ORF2 from five pairs of serum and fecal samples. All strains were subtype b in genotype 3 (HEV-3b) but separated into different clusters. We determined the entire genomesequence of HEV-3b strain WB0567 using a fecal specimen and isolated this strain by cell culture using PLC/PRF/5 cells. Eleven nucleotide mutations had occurred during virus replication.These results suggest that HEV-3b circulated uniformly among wild boars in Japan. Direct sequencing using a suspected animal’s samples is indispensable for predicting original HEV nucleotidesequences.
Keywords: hepatitis E virus (HEV), genotype 3 HEV, subtype, wild boar, zoonosis
Hepatitis E virus (HEV), a causative agent of human hepatitis E, is a single-stranded positive-sense RNA virus that is transmitted primarily by fecal–oral routes through contaminated drinkingwater [2]. HEV is classified in the family Hepeviridae, which includes two subfamilies: Orthohepevirinae andParahepevirinae (ictv.global/taxonomy). The subfamily Orthohepevirinae includes at least four genera: Paslahepevirus,Rocahepevirus, Chirohepevirus, and Avihepevirus [13]. The genus Paslahepevirus isclassified into two species, alci and balayani, and the species balayani includes eight viral genotypes, HEV-1 to HEV-8 [16]. Several studies have confirmed that various animal species are susceptible to HEV, and it is noteworthy that HEV is transmitted to humans from swine, wild boars, rabbits,camels, and rats and causes type E hepatitis [1, 4, 5, 17].
In Japan, HEV-3, -4, -5, and -6 have been detected in wild boars [18, 19], suggesting that this animal is animportant reservoir of HEV. In fact, several hepatitis E cases are known to be epidemiologically linked to the consumption of undercooked wild boar meat, liver, or viscera [10]. We reported direct evidence of HEV transmission from a wild boar to humans [5]. However, the current status of HEV infection in wildboars in Japan remains unclear.
From 2005 to 2006, a total of 162 paired serum and fecal samples were collected from wild boars in Japan. The sample pairs were from Nagasaki (n=60), Shizuoka (n=29), Kumamoto and Kochi (n=15),Hiroshima (n=14), Chiba and Hyogo (n=12), and Shimane Prefecture (n=5). During the same period, 17 serum samples were collected exclusively from Nagasaki Prefecture. All wild boars were capturedby hunters for purpose of expulsion. The blood and fecal specimens were collected and sent immediately to the National Institute of Infectious Diseases Japan in a refrigerated state, and thesample preparations were done as promptly as possible after receiving the specimens. The serum samples were separated by centrifugation at 2,500 g for 30 min at 4°C. Three gramsof the fecal specimens were diluted with 30 mL of 10 mM phosphate-buffered saline (PBS) to prepare a 10% (w/v) suspension as described previously [21]. Allof the serum samples and fecal suspensions were stored at −80°C until use.
The serum samples were diluted to 1:200, and the detection of the antibodies was performed by an enzyme-linked immunosorbent assay (ELISA) using HEV-like particles (HEV-LPs) as the antigen[6] and a peroxidase-labeled affinity purified goat anti-swine IgG (H+L) antibody (KPL, Guilford, UK) at 1:4,000 dilution. The cutoff value of the ELISAwas determined by using a total of 43 serum samples that were negative for anti-HEV IgG antibodies and had been collected from wild boars in Japan’s Ibaraki Prefecture in 2013–2014 [12]. The optical density (OD) values of the anti-HEV IgG antibodies ranged from 0.012 to 0.147; the mean OD value was 0.044 with a standard deviation (SD) of0.041. The cutoff value was thus calculated as 0.167 on the basis of the mean OD values plus 3 times the SD (0.044 + 3 × 0.041).
The anti-HEV IgG antibody levels in the 179 serum samples ranged from 0.021 to 3.246, and 39 of the 179 samples (21.8%) were positive (Table 1). Theanti-HEV IgG antibody-positive rate in the prefectures was 37.9% (11/29) in Shizuoka, 23.4% (18/77) in Nagasaki, 25% (3/12) in Hyogo, 20% (3/15) in Kumamoto, 20% (1/5) in Shimane, 8.3% (1/12) inChiba, 7.1% (1/14) in Hiroshima, and 6.7% (1/15) in Kochi (Table 1). In Shizuoka Prefecture, the wild boars were captured in two areas, Hamamatsu(n=10) and Izu Peninsula (n=19); the antibody-positive rate was 52.6% (10/19) in Izu Peninsula, whereas no antibody was detected in the animals captured in Hamamatsu. Hamamatsu is located in thewestern part of Shizuoka Prefecture, and Izu is located in the eastern part. The distance between the two areas is approx. 170 km. These results suggested that the infection status of wild boarsto HEV differs greatly depending on the area.
Table 1.
Detection of anti-hepatitis E virus (HEV) IgG antibody and HEV RNA
| Prefecture | Serum samples | Fecal specimens | |||||
|---|---|---|---|---|---|---|---|
| No. of samples | Anti-HEV IgG positive (%) | HEV RNA positive (%)* | Genotype** | No. of samples | HEV RNA positive (%) | Genotype | |
| Nagasaki | 77 | 18 (23.4) | 2 (2.6) | G3 (1) | 60 | 1 (1.7) | G3 (1) |
| Shizuoka | 29 | 11 (37.9) | 4 (13.8) | G3 (2) | 29 | 2 (6.9) | G3 (2) |
| Kumamoto | 15 | 3 (20.0) | 1 (6.7) | G3 (1) | 15 | 1 (6.7) | G3 (1) |
| Kochi | 15 | 1 (6.7) | 1 (6.7) | Non*** | 15 | 0 (0) | Non |
| Hiroshima | 14 | 1 (7.1) | 1 (7.1) | G3 (1) | 14 | 1 (7.1) | G3 (1) |
| Chiba | 12 | 1 (8.3) | 1 (8.3) | Non | 12 | 0 (0) | Non |
| Hyogo | 12 | 3 (25) | 1 (8.3) | Non | 12 | 0 (0) | Non |
| Shimane | 5 | 1 (20) | 0 (0) | Non | 5 | 0 (0) | Non |
| Total | 179 | 39 (21.8) | 11 (6.1) | 162 | 5 (3.1) | ||
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*HEV RNA were detected by RT-qPCR. **HEV RNA were detected by RT-PCR and the partial nucleotide sequences were analyzed. *** HEV RNA were detected by RT-qPCR, but the genotype could notbe determined.
All of the serum and fecal specimens were used for the detection and quantification of HEV RNA by a one-step quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR)[3], and a 10-fold serial dilution of HEV-3 (107 to 101 copies) was used as the standard for the quantitation of the copy numbers[21]. As shown in Table 1, 6.1% (11/179) of the serum samples and 3.1% (5/162) of the fecal specimens werepositive. The copy numbers of the virus RNA in the serum samples ranged from 4.51 × 102 to 1.49 × 105 copies/mL, and those in the fecal specimens ranged from 4.29 ×105 to 2.70 × 108 copies/g. All HEV RNA-positive samples were further amplified by RT-PCR [11], and 412 base pairs (bp) in openreading frame 2 (ORF2) were identified in five pairs of the specimens. The nucleotide sequences in each pair were identical (GenBank accession nos. LC770331 and LC774729 to LC774732) (Fig. 1a).
Fig. 1.
The phylogenetic analysis based on the partial ORF2 (412 bp) (a) and the phylogenetic analysis based on the entire genome (c). The former analysis included fiveHEV-3b strains detected in this study (highlighted in bold), and 40 strains of HEV-3b and 62 strains of other HEV-3 subtypes. The latter analysis was performed toconstellate the WB0567 strain with 26 strains of HEV-3b and 62 strains of other HEV-3 subtypes. The accession number, host, country, collection date and strain name of each strain wereshown, and the unclear information such as collection date or host was indicated as xxxx (a, c). The phylogenetic trees were generated using the MaximumLikelihood method based on the General Time Reversible model implemented in the MEGA6 software package. Bootstrap values were calculated through 1,000 replicate trials, providingstatistical support for the tree branches. The genetic distance is indicated at the bottom, and the percentage bootstrap support is displayed at each node, denoting support values of ≥70%.The geographical distribution of HEV RNA detected in wild boars is shown in (b).
The strains WB0534 (LC774729) and WB0567 (LC770331) were from Shizuoka, WB0544 (LC774730) was from Hiroshima, WB0565 (LC774731) was from Kumamoto, and WB0613 (LC774732) was from Nagasaki (Fig. 1b). Phylogenetic analyses indicated that all five strains belonged to HEV-3, subtype b (HEV-3b), but they separated into different clusters. TheWB0534 strain shares 98.3% nucleotide identity with strain wbjsi-05-2-L2 (AB605214) that was detected in a wild boar in the same prefecture, i.e., Shizuoka, in 2005 [15], suggesting that similar HEV-3b was circulating in wild boars in this area. WB0534 shares 89.3% nucleotide identity with WB0567 although these strains were from boars thatwere captured in the Shirahama area of Izu, suggesting that multiple HEV strains circulated in wild boars in the same area. Strain WB0544 detected in Hiroshima Prefecture shares 99.8% nucleotideidentity with a strain detected in a blood donor in Chiba Prefecture in Japan in 2003, i.e., JRC-HE1 (AB434144) [14]. Strain WB0565 detected in KumamotoPrefecture shares 99.8% nucleotide identity with a strain HRC-HE28 (AB670964) detected in 2005 in a blood donor in the city of Sapporo, Hokkaido Prefecture: The HEV-3b that exists in wild boarshas high genome identity with the HEV-3b detected in humans in different areas, suggesting that zoonotic HEV infection from wild boars has occurred frequently. In contrast, strain WB0613detected in Nagasaki shares <91.3% nucleotide identity with other published HEV strains, suggesting that an indigenous HEV-3b circulated in Nagasaki Prefecture.
The entire genome sequence of strain WB0567 was analyzed by next-generation sequencing (NGS) [7]. This strain (accession no. LC770331) consists of 7,227nucleotides and an undetermined length of poly (A) tail. The 5’ terminal untranslated region (5’UTR) is comprised of 26 nucleotides (nucleotide sequence nos. 1–26) and the 3’UTR has 72nucleotides (7,156–7,227). WB0567 contains three ORFs: ORF1 (27–5,138 nt) with 1,703 amino acids (aa), ORF3 (5,135–5,503nt) with 122 aa, and ORF2 (5,173–7,155 nt) with 660 aa.
A phylogenetic analysis of HEV based on the entire genome also indicated that WB0567 belongs to HEV-3b, forming a cluster with five strains: JHS-Kan06L (LC406523), JMT-Chiba08L (LC406572),swEJM1201802SC (LC704569), swEJM2100729FC (LC704570) and swJ570 (AB037912) (Fig. 1c). However, BLAST analyses showed that WB0567 shares 91.8% nucleotidesequence identity with the strain JMT-Chiba08L detected in a patient in Chiba Prefecture, 91.0% identity with the strain JHS-Kan06L detected in a patient in Kanagawa Prefecture (LC406572), and92.1%, 91.3% and 90.6% identity with strains swEJM1201802SC, swEJM2100729FC and swj570 detected in swine in Japan [20] (Fig. 1c).
In an attempt to isolate the HEV-3b strain by cell culture, we used the fecal suspension from wild boar WB0567 containing 2.70 × 107 copies/mL of the virus RNA to inoculate humanhepatocarcinoma cell line, PLC/PRF/5 (JCRB0406), which is susceptible to HEV infection and used for HEV isolation [9]. The virus RNA and the capsid proteinwere monitored by RT-qPCR and ELISA, respectively [9] (Fig. 2). The virus RNA was detected in the culture supernatant on day 4 post-inoculation (p.i.) at 3.2 × 103 copies/mL; similar viral RNA copy numbers were observed until day 24 p.i.and then the numbers gradually increased to 1.95 × 108 copies/mL on day 72 p.i. A peak was reached on day 80 p.i. at 5.92 × 108 copies/mL, and then a plateau occurredbetween 3.31 × 108 copies/mL and 5.90 × 108 copies/mL until day 100 p.i. (Fig. 2a). The capsid protein was detected on day 48 p.i.with an OD value of 0.189, then increased gradually and reached a peak on day 80 p.i. with an OD value of 1.332 (Fig. 2b). We named the virus recoveredfrom the supernatants of WB0567-inoculated cells “WB0567c1”.
Fig. 2.
Replication of strain WB0567 in PLC/PRF/5 cells. The confluent PLC/PRF/5 cells were dispersed by trypsinization, and 2 × 105 cells were cultured in a 25-cm2 tissueculture flask for 24 hr. The cells were then washed with PBS, and 1 mL of 10% wild boar fecal suspension was inoculated onto PLC/PRF/5 cells. After adsorption at 37°C for 1 hr, the cellswere washed three times with PBS, followed by further incubation with 10 mL of the maintenance medium at 36°C [8]. WB0567c1 was recovered from theculture supernatants of WB0567-inoculated cells (○). WB0567c2 was recovered from the culture supernatants of WB0567c1-inoculated cells (Δ). The culture supernatants were collected every 4days and used for the detection of the virus RNA by RT-qPCR (a) and capsid antigen by ELISA (b). Dotted lines: The cut-off value for thedetection of HEV capsid protein was 0.15. The minimum OD value of the positive samples is blackened.
To examine the infectivity of WB0567c1, we used the cell culture supernatants collected on day 80 p.i. to inoculate PLC/PRF/5 cells. Extensive virus growth was observed by an earlier detectionof both virus RNA and the capsid antigen in the culture medium (Fig. 2a, 2b). The virus RNA was detected in the culture supernatant on day 4 p.i. at6.20 × 103 copies/mL and increased to 1.24 × 108 copies/mL on day 44 p.i.; it then showed a plateau above 3.43 × 108 copies/mL (Fig. 2a). The capsid protein was detected on day 12 p.i. with an OD value of 0.155, then increased and reached a peak on day 60 p.i. with the OD value 1.328 (Fig. 2b). We named the virus recovered from the supernatants of WB0567c1-infected cells “WB0567c2”. These results demonstrated that HEV-3b strain WB0567isolated from a wild boar efficiently replicated in PLC/PRF/5 cells.
Based on the NGS analyses, the entire genome sequences of WB0567c1 (LC774371) and WB0567c2 appeared to be identical, but a total of 11 nucleotides differed from that of the original strainWB0567, suggesting that nucleotide mutations occurred during virus replication in PLC/PRF/5 cells. Eight nucleotide mutations occurred in ORF1, leading to three amino acid changes (Ser509Leu,His832Tyr, and Gln1352Arg). Two nucleotide mutations appeared in ORF2 (nt 5,703 and 6,147), and another nucleotide mutation in 3’UTR (nt 7,186) had no amino acid change in ORF2 (Table 2). These results suggested that (i) unexpected mutations occurred frequently and (ii) it is quite difficult to obtain the original sequences afterpropagation in cell culture, although PLC/PRF/5 cells are a convenient tool for the isolation of HEV.
Table 2. Nucleotide and amino acid changes in the WB0567c1 strain
| Nucleotide changes | Amino acid changes | |||||
|---|---|---|---|---|---|---|
| aPosition | WA0567 | WB0567c1 | bPosition | ORF1 | ORF2 | ORF3 |
| 1,552 | C | T | 509 | Ser/Leu | ||
| 1,865 | G | T | ||||
| 2,520 | C | T | 832 | His/Tyr | ||
| 2,645 | C | T | ||||
| 3,635 | C | T | ||||
| 4,064 | T | C | ||||
| 4,081 | A | G | 1,352 | Gln/Arg | ||
| 4,842 | C | T | ||||
| 5,703 | T | A | ||||
| 6,147 | C | T | ||||
| 7,186 | C | T | ||||
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aNucleotide positions in the genomic RNA. bAmino acid positions in each open reading frame (ORF).
In the present study, we observed a high prevalence of HEV infection and regional differences in HEV sero-prevalence in wild boars in Japan. The HEV strains belonging to subtype 3b wereidentified in at least four of the eight prefectures from which the wild boars were captured, suggesting that HEV-3b is a major subtype of HEV-3 circulating in wild boars in Japan. BLASTanalyses showed that the WB0567 shares <92.1% nucleotide sequence identity with all HEV-3b strains insolated from swine, suggesting that HEV strains unique to the wild boars are circulating.As the HEV-3b strain, WB0567 was isolated with the use of PLC/PRF/5 cells, and this cell culture system might therefore be useful for studies of the mechanisms underlying the replication andinfection of HEV. However, we should emphasize that direct sequencing using materials derived from suspected animals is indispensable in predicting original HEV nucleotide sequences.
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
Acknowledgments
We thank Miyuki Oizumi for the technical assistance and Naokazu Takeda for revising the manuscript. This research was supported by grants from the Research Program on Hepatitis (nos.JP23fk0210109, JP23fk0210132, JP23wm0225009) from the Japan Agency for Medical Research and Development (AMED).
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