Generation of a Bactrian camel hepatitis E virus by a reverse genetics system

Abstract

Bactrian camel hepatitis E virus (HEV) is a novel HEV belonging to genotype 8 (HEV-8) in the Orthohepevirus A species of the genus Hepevirus in the family Hepeviridae. HEV-8 cross-transmits to cynomolgus monkeys and has a potential risk for zoonotic infection. Until now, neither a cell-culture system to grow the virus nor a reverse genetics system to generate the virus has been developed. To generate replication-competent HEV-8 and to establish a cell-culture system, we synthesized capped genomic HEV-8 RNAs by in vitro transcription and used them to transfect into PLC/PRF/5 cells. A HEV-8 strain, HEV-8M2, was recovered from the capped HEV-8 RNA–transfected cell-culture supernatants and subsequently passaged in the cells, demonstrating that PLC/PRF/5 cells were capable of supporting the replication of the HEV-8, and that a cell-culture system for HEV-8 was successfully established. In addition to PLC/PRF/5 cells, A549 and Caco-2 cells appeared to be competent for the replication, but HepG2 C3/A, Vero, Hela S3, HEp-2C, 293T and GL37 cells were incompetent. The HEV-8M2 strain was capable of infecting cynomolgus monkeys by an intravenous inoculation, indicating that HEV-8 was infectious and again carried a risk for zoonotic infection. In contrast, HEV-8 did not infect nude rats and BALB/c nude mice, suggesting that the reservoir of HEV-8 was limited. In addition, the replication of the HEV-8M2 strain was efficiently abrogated by ribavirin but not by favipiravir, suggesting that ribavirin is a drug candidate for therapeutic treatment of HEV-8-induced hepatitis. The infectious HEV-8 produced by a reverse genetics system would be useful to elucidate the mechanisms of HEV replication and the pathogenesis of type E hepatitis.

A Cross-Species Transmission of a Camel-Derived Genotype 8 Hepatitis E Virus to Rabbits

“…Cynomolgus monkeys are susceptible to HEV-5, HEV-7, and HEV-8, but the costs accompanying their use are high, and recent studies indicated that most of the imported cynomolgus monkeys have been exposed to HEV infection, suggesting that the source of the monkey is limited [14]. Our research has confirmed that rats and mice are not susceptible to HEV-8 infection [9]. A tractable small animal remains necessary for this line of research.…”

“…All of these HEV strains were produced by a reverse genetics system using a human hepatocarcinoma cell line, PLC/PRF/5 cells (JCRB0406) (JCRB Cell Bank, Osaka, Japan), containing both "quasi-enveloped viruses" and "naked viruses" and that induced infection in cynomolgus monkeys or rabbits. [6,7,9,17]. The culture supernatants of the HEV-infected PLC/PRF/5 cells were clarified by centrifugation at 10,000× g for 30 min and then passed through a 0.45 µm membrane filter (Millipore, Bedford, MA, USA).…”

“…N-terminal 111 aa-truncated ORF2 was amplified by PCR using a plasmid, pUC57-T7HEV-8K, containing the complete genome of HEV-8 (KX387866) as the template [9]. The PCR product was digested with BamHI and XbaI and then ligated with a baculovirus transfer vector, pVL1393 (Pharmingen, San Diego, CA, USA) to yield the plasmid pVL1393-G8n111ORF2.…”

“…The species Orthohepevirus A includes eight genotypes designated genotypes 1 to 8 (HEV-1 to HEV-8) [3][4][5]. Although most cases of hepatitis E have been caused by HEV-1 to HEV-4, novel HEVs including HEV-5, HEV-7, and HEV-8 are known to cross-transmit to non-human primates such as cynomolgus monkeys, raising a potential risk of zoonotic infection [6][7][8][9]. In fact, a human case of hepatitis E due to HEV-7 infection was reported in 2016 [10].…”

“…HEV-5, HEV-7, and HEV-8 were first detected in wild boar, dromedary camels, and Bactrian camels, respectively [11][12][13]. Since replication-competent strains of these viruses were successfully produced by a reverse genetics system using a human hepatocarcinoma cell line, PLC/PRF/5 [6,7,9], the mechanism of the replication of these HEVs might be elucidated using the cell culture ex vivo. The original reservoirs of HEV-5, HEV-7, and HEV-8 are large animals that are not easily used as animal models, especially to understand the pathogenicity of HEV.…”