Rat Hepatitis E Virus as Cause of Persistent Hepatitis after Liver Transplant

All hepatitis E virus (HEV) variants reported to infect humans belong to the species Orthohepevirus A (HEV-A). The zoonotic potential of the species Orthohepevirus C (HEV-C), which circulates in rats and is highly divergent from HEV-A, is unknown. We report a liver transplant recipient with hepatitis caused by HEV-C infection. We detected HEV-C RNA in multiple clinical samples and HEV-C antigen in the liver. The complete genome of the HEV-C isolate had 93.7% nt similarity to an HEV-C strain from Vietnam. The patient had preexisting HEV antibodies, which were not protective against HEV-C infection. Ribavirin was an effective treatment, resulting in resolution of hepatitis and clearance of HEV-C viremia. Testing for this zoonotic virus should be performed for immunocompromised and immunocompetent patients with unexplained hepatitis because routine hepatitis E diagnostic tests may miss HEV-C infection. HEV-C is also a potential threat to the blood product supply.

The PCR mixture (25 L) contained cDNA, 1 PCR buffer II (10 mM ], 50 mM KCl), 2 mM MgCl2, 200 M of each dNTP, 1 M forward and reverse primers and 1.0 U of Taq polymerase (Applied Biosystems, Foster City, CA, USA). PCR was performed using an automated thermocycler (Applied Biosystems) with a hot start at 95C for 10 min, followed by 40 cycles of 94C for 1 min, 55C for 1 min, and 72C for 1 min and a final extension at 72C for 10 min. The PCR products were detected by agarose gel electrophoresis (staining gel with ethidium bromide, followed by visualization under UV light).

HEV Complete Genome Sequencing
PCR amplicon of the pan-Orthohepevirus RT-PCR assay was extracted using the QIAquick gel extraction kit (Qiagen). Both strands of the PCR product were sequenced twice with an ABI Prism 3130xl DNA Analyzer (Applied Biosystems) using primers listed in Technical Appendix Table 1. As the 235 bp RNA-dependent RNA polymerase (RdRp) sequences of HEV isolates obtained from patient specimens clustered with rat HEV-C strains, degenerate primers for complete genome amplification were designed by multiple alignment of the rat HEV-C genomes available in GenBank as per Technical Appendix Table 2. RNA extracted from the patient's stool (containing HEV isolate LCK-3110) was converted to cDNA by a combined random-priming and oligo(dT) priming strategy; this cDNA was used as the template for complete genome sequencing. The 5′ end of LCK-3110 was confirmed by rapid amplification of cDNA ends using Terminal Deoxynucleotidyl Transferase, recombinant (Invitrogen). Sequences were assembled and manually edited to produce final sequences of the viral genomes by BioEdit version 7.2.5 (NC State University, Raleigh, NC, USA).
Amplified genes were cloned into the Nde I and Xol I sites of bacterial expression vector pETH in frame and downstream of the series of 6 histidine residues. The recombinant 239 aa HEV-A and HEV-C proteins were expressed in Escherichia coli and purified by using the Ninitrilotriacetic acid affinity chromatography assay (Qiagen) according to manufacturer instructions. The HEV-A and HEV-C proteins had a 219 aa overlapping fragment and shared 52% sequence homology. The sequence alignment of the 2 proteins is as follows:  For the HEV-A and HEV-C in-house ELISAs, 96-well microwell plates (Costar, Corning, NY, USA) were coated with purified recombinant HEV-A and HEV-C proteins. 100 L of serum from three asymptomatic blood donors who tested negative in the Wantai HEV IgG ELISA and both HEV-A and HEV-C IgG Western blots was diluted 1:500 in 0.1% bovine serum albumin (BSA). Diluted serum was added to the ELISA plates (2 replicates) and incubated at 37C for 1 h. After a washing step, goat anti-human-horse radish peroxidase (100 L/ well) was added to plates followed by incubation at 37C for 30 min. After washing, tetramethylbenzidate substrate was added. The reaction was stopped after 10 min by addition of 0.3 N sulphuric acid.
Plates were examined in an ELISA plate reader at 450 nm. The mean optical density (OD) of these negative control sera was 0.152 (range 0·132-0·171; SD  0.017) for HEV-C ELISA and 0.178 (range 0.169-0.187; SD  0·008). Based on these findings, a tentative ELISA cutoff of 0.3 was designated. As subsequent ELISA experiments were quantal measurements using serial dilutions of patient serum, a precise cutoff is not necessary. For the ELISA experiment, patient serum was serially diluted 2-fold from 1:500 to 1:64,000 in BSA followed by HEV-A and HEV-C IgG ELISA as described above. The OD values were plotted against serum dilution. The IgG titer of a serum specimen was defined as the reciprocal of the highest dilution that gave an OD value above the cutoff. Using the tentative cutoff of 0.3, a serum sample would be considered to be specifically reactive against a particular HEV antigen (i.e., HEV-A or HEV-C) if there was a 4-fold difference in IgG titers between the 2 ELISAs. For example, if the IgG titer above the cutoff line was 4,000 by HEV-A ELISA and 8,000 by HEV-C ELISA, the serum would not be considered to be more reactive against 1 HEV antigen over the other. Conversely, if the HEV-A ELISA IgG titer was 4,000 and HEV-C ELISA IgG titer was 16,000, then the serum would be considered to be more reactive against HEV-C than HEV-A.

Cell Culture
The patient's stool was diluted in phosphate buffered saline (PBS) and filtered to produce a 10% suspension. The suspension was further diluted 1:10 (for A549 and Huh-7) and 1:100 (for Caco-2) and 300 L of suspension was used to inoculate cells in 12-well plates at an estimated multiplicity-of-infection of 5 HEV genome equivalents/ cell. Inoculum was removed after 1 h and replaced with minimum essential medium supplemented with 1% fetal calf serum. Cell lines were incubated at 37C and were examined for cytopathic effect (CPE) daily. On day 3 and day 5, 250 L of supernatant was collected and replenished with fresh medium. Cell monolayers were maintained for 7 d before harvesting of cell lysate. A549 cell monolayers were inoculated in chamber wells for immunohistochemical staining.

Immunohistochemical and Immunofluorescence Staining
Immunofluorescence staining of infected and uninfected A549, Huh-7, and Caco-2 7-day De-paraffinized liver tissue sections were treated with antigen unmasking buffer (Vector Laboratories, Burlingame, CA, USA) and hydrogen peroxide block. Slides were then incubated with primary reagent (either cross-reactive monoclonal antibody or bovine serum albumin) overnight at 4C. After rinsing, slides were incubated with biotin conjugated goat anti-mouse IgG at room temperature for 30 min. Rinsed slides were then incubated with HRP-streptavidin followed by color development using 3, 3′-diaminobenzidine (Vector Laboratories). Liver tissue section slides were counterstained with Gill's hematoxylin and examined using light microscopy.

Description of LCK-3110 Complete Genome
Predicted genomic organization of LCK-3110 was similar to other rat HEV isolates: from 5′ to 3′ ends, it consists of a 5′-untranslated region at nucleotide positions 1-10, ORF1 at nt 11- was only 47.7%, which was even lower than the overall ORF2 sequence amino acid homology.
In-silico epitope analysis of the target residue sites of the 8C11 HEV-A neutralizing monoclonal antibody showed that only 1 out of 6 residues were conserved between LCK-3110 and the Xinjiang strain with a serine to glutamic acid substitution at the critical host-specificity defining residue aa 497 (HEV-A genotype 1 numbering).