Announcements

Authors: Putu Prathiwi Primadharsini, Masaharu Takahashi, Tsutomu Nishizawa, Yukihiro Sato, Shigeo Nagashima, Kazumoto Murata and Hiroaki Okamoto

Published in the Special Issue “Hepatitis E: Molecular Virology, Pathogenesis, and Treatment” of Viruses on 24 May 2024

Hepatitis E virus (HEV), the causative agent of hepatitis E, represents a significant but largely understudied human pathogen. According to the 2023 World Health Organization report, HEV accounts for approximately 20 million infections worldwide annually, resulting in around 44,000 hepatitis E-related deaths in 2015. HEV primarily spreads through the fecal–oral route, typically via contaminated drinking water in developing countries and through the consumption of undercooked or raw animal meat in industrialized nations ⁽¹⁾. HEV belongs to the family Hepeviridae, whereby genotypes 1 and 2 (HEV-1 and HEV-2) within the Paslahepevirus balayani species exclusively infect humans (https://ictv.global/report/chapter/hepeviridae/hepeviridae), while HEV-3 and HEV-4 infect humans as well as several other animal species. HEV-5 and HEV-6 are found in wild boars, while HEV-7 and HEV-8 infect camels. HEV-5, HEV-7, and HEV-8 are reported to infect humans or have the potential to do so ^(2-4). However, such data are unavailable for HEV-6, and cell culture and reverse genetics systems have not been established for HEV-6 thus far due to the low viral load of two previously identified HEV-6 strains (GenBank accession numbers AB602441 and AB858243).

The primary aim of this study was to identify a new HEV-6 strain with a high viral load from wild boars in Japan, characterize its genetic makeup, and determine whether it could infect human-derived cells to investigate the zoonotic potential of HEV-6. The secondary goal was to construct a full-length cDNA clone and evaluate the effectiveness of ribavirin as a treatment.

This study involved collecting serum and liver specimens from eight wild boars captured in Hyogo Prefecture, Japan, in 2023 and analyzing them for anti-HEV IgG antibodies and HEV RNA. A liver homogenate from an HEV-positive boar (wbJHG_23) was used as an inoculum for human-derived cancer cell lines (PLC/PRF/5 and A549 1-1H8, a subclone of A549). The ability of the virus to infect and propagate in these cells was monitored by measuring HEV RNA titers over time. RNA was extracted from culture supernatants, and a full-length infectious cDNA clone was constructed using reverse genetics. The genetic diversity of the wbJHG_23 strain was analyzed by sequencing and comparing it with known HEV genotypes (HEV-1 to HEV-8). The effectiveness of ribavirin in inhibiting HEV-6 replication was assessed by treating infected cell cultures with various concentrations of the drug and monitoring viral RNA levels. The infectivity of the virus and the expression of HEV ORF2 (capsid) and ORF3 (a multifunctional protein involved in virion assembly and release) proteins were confirmed through Western blotting and immunofluorescence assays.

This study revealed that out of the eight wild boars tested, only one (wbJHG_23) was positive for anti-HEV IgG antibodies and HEV RNA, indicating an active HEV infection. The wbJHG_23 strain was identified as HEV-6, sharing 82.5%­83.0% of nucleotide sequence identity with known HEV-6 strains and a lower similarity to other HEV genotypes. Phylogenetic analysis confirmed that wbJHG_23 clustered closely with two previously reported HEV-6 strains. The wbJHG_23 strain successfully infected and replicated in both PLC/PRF/5 and A549 1-1H8 cell lines, with viral RNA titers reaching over 10^8 copies/ml. In addition, Western blotting and immunofluorescence assays confirmed the expression of HEV ORF2 and ORF3 proteins in infected cells, suggesting that HEV-6 has the potential for zoonotic infection. Ribavirin treatment effectively inhibited HEV-6 replication in a dose-dependent manner in both cell lines. The viral RNA titers were significantly reduced in treated cultures compared to untreated controls. A full-length infectious cDNA clone of the wbJHG_23 strain was successfully constructed. RNA transcripts from this clone were transfected into PLC/PRF/5 cells, resulting in the production of infectious virus progeny, demonstrating the functionality of the cDNA clone (Figure 1).

Figure 1. Comprehensive workflow for the generation and analysis of HEV-6 (wbJHG_23) in PLC/PRF/5 cells.

In conclusion, this study successfully identified and characterized a new strain of HEV-6 (wbJHG_23) from a wild boar in Japan, demonstrating its ability to infect human-derived cell lines with high replication efficiency and suggesting the zoonotic potential of HEV-6. This study also demonstrated its susceptibility to ribavirin treatment. The construction of an infectious cDNA clone provides a valuable tool for future research on HEV-6, particularly in studying its zoonotic potential and developing antiviral strategies. The confirmation of the suggested zoonotic transmission of HEV-6 necessitates further investigation through infection experiments involving non-human primates. This study updates previous reports ^(5-7) on the establishment of cell culture and reverse genetics systems, as well as the zoonotic potential of eight genotypes of HEVs (HEV-1 to HEV-8) within the species Paslahepevirus balayani (Table 1).

Table 1. Establishment of cell culture and reverse genetics systems, and cross-species transmission of Hepatitis E virus within the species Paslahepevirus balayani.

For more details on the experiments and results, please refer to our paper, which can be found at https://doi.org/10.3390/v16060842.

References

1. Wang B, Meng XJ. Hepatitis E virus: host tropism and zoonotic infection. Curr Opin Microbiol. 2021 Feb;59:8-15. doi: 10.1016/j.mib.2020.07.004.PMID: 32810801.
2. Lee GH, Tan BH, Teo EC, Lim SG, Dan YY, Wee A, Aw PP, Zhu Y, Hibberd ML, Tan CK, Purdy MA, Teo CG. Chronic Infection With Camelid Hepatitis E Virus in a Liver Transplant Recipient Who Regularly Consumes Camel Meat and Milk. Gastroenterology. 2016 Feb;150(2):355-7.e3. doi: 10.1053/j.gastro.2015.10.048. PMID: 26551551.
3. Wang L, Teng JLL, Lau SKP, Sridhar S, Fu H, Gong W, Li M, Xu Q, He Y, Zhuang H, Woo PCY, Wang L. Transmission of a Novel Genotype of Hepatitis E Virus from Bactrian Camels to Cynomolgus Macaques. J Virol. 2019 Mar 21;93(7):e02014-18. doi: 10.1128/JVI.02014-18. PMID: 30700602.
4. Li TC, Bai H, Yoshizaki S, Ami Y, Suzaki Y, Doan YH, Takahashi K, Mishiro S, Takeda N, Wakita T. Genotype 5 Hepatitis E Virus Produced by a Reverse Genetics System Has the Potential for Zoonotic Infection. Hepatol Commun. 2018 Nov 30;3(1):160-172. doi: 10.1002/hep4.1288. PMID: 30620002.
5. Primadharsini PP, Nagashima S, Okamoto H. Mechanism of Cross-Species Transmission, Adaptive Evolution and Pathogenesis of Hepatitis E Virus. Viruses. 2021 May 14;13(5):909. doi: 10.3390/v13050909. PMID: 34069006.
6. Zhang W, Ami Y, Suzaki Y, Doan YH, Takeda N, Muramatsu M, Li TC. Generation of a Bactrian camel hepatitis E virus by a reverse genetics system. J Gen Virol. 2021 Jul;102(7). doi: 10.1099/jgv.0.001618. PMID: 34242156.
7. Scholz J, Falkenhagen A, Bock CT, Johne R. Reverse genetics approaches for hepatitis E virus and related viruses. Curr Opin Virol. 2020 Oct;44:121-128. doi: 10.1016/j.coviro.2020.07.004. PMID: 32818718.