Persistent infection with a rabbit hepatitis E virus created by a reverse genetics system

Abstract

Rabbit hepatitis E virus (HEV) is a novel zoonotic infectious agent. Although a cell culture system to grow the virus has been established, there is currently no reverse genetics system for generating the virus. In this study, capped genomic rabbit HEV RNAs generated by in vitro transcription were transfected into PLC/PRF/5 cells, and the recovered viruses were subsequently passaged in the cells. The cell culture supernatant was capable of infecting rabbits negative for anti-HEV antibody by intravenous and oral inoculation, indicating that rabbit HEV generated by the reverse genetics system is infectious. Genome-wide analyses indicated that no nucleotide sequence change occurred in the virus genomes that were recovered from the cell culture supernatant after transfection and passaged one time or in the virus genomes recovered from faecal specimens of the infected rabbits. Ribavirin, a broad-spectrum anti-viral inhibitor, efficiently abrogated virus replication ex vivo and transiently suppressed the virus growth in the virus-infected rabbits, suggesting that this reagent is a candidate for therapeutic treatment. In addition, transmission of rabbit HEV to rabbits caused persistent infection, suggesting that the virus-infected rabbit could be an animal model for virus-induced hepatitis. The infectious rabbit HEV produced by a reverse genetics system would be useful to elucidate the mechanisms of HEV replication and the pathogenesis of viral hepatitis.

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

“…Reverse genetics systems are a powerful tool, and have been widely used to produce infectious HEVs, including HEV-1,–3, -4,–5, −7 and rat HEV [19, 30–32, 34, 35]. We selected two HEV-8 strains, and the capped genome RNAs, HEV-8M2 RNA and HEV-8K RNA , were synthesized in vitro and used to transfect PLC/PRF/5 cells.…”

“…The HEV-8 capsid protein was detected by an antigen-capture ELISA as described previously with slight modifications [19,30,31]. Briefly, a 96-well microplate was coated with 1 : 2000 diluted rabbit anti-HEV-1 VLPs serum, and 100 µl of the cell-culture supernatant was added.…”

“…ALT values in the monkey sera were monitored weekly using a Fuji Dri-Chem Slide GPT/ALT-PIII kit (Fujifilm, Saitama, Japan). The geometric mean litres of ALT over the preinoculation period were defined as normal ALT, and a twofold or greater increase at the peak was considered a sign of hepatitis [31].…”

Immunogenicity and Antigenicity of Rabbit Hepatitis E Virus-Like Particles Produced by Recombinant Baculoviruses

“…Two types of the truncated ORF2s of rabbit HEV were amplified by a polymerase chain reaction (PCR) using a plasmid, pUC57-T7RHEV-F, as a template containing the complete genome of a rabbit HEV strain (JQ013791) [29]. An N-terminal 13 aa-truncated ORF2 was amplified with a forward primer RFN13 (5 -AGGATCCATGCTGCCTATGCTGCCCGCGCC A-3 ) and a reverse primer RCR (5 -CTCTAGATTAAGACTCCCGGGTTTTACCTA-3 ), whereas an N-terminal 111-aa-truncated ORF2 was amplified with a forward primer RFN111 (5 -AGGATCCATGGCCGTTTCACCAGCCCCTGACA-3 ) and the same reverse primer.…”

“…Therefore, the diagnosis and vaccine development for rabbit HEV stand in need of urgent attention. Although a cell culture system to grow the virus has been established, it is difficult to obtain the large amounts of antigen required for diagnosis and vaccine development using this system [28,29].…”