The rabbit HEV strain (CHN-BJ-R14) used in this study was originally recovered from the feces of a farmed Rex Rabbit in the suburbs of Beijing in 2011. The fecal sample was diluted in phosphate-buffered saline (PBS) (pH 7.4) containing 1% bovine serum albumin to make a 10% (wt/vol) suspension. The clarified suspension was subsequently filtered through 0.45-μm and 0.22-μm filters. Titers of the rabbit HEV inoculum were determined by a semiquantitative nested reverse transcription PCR (RT-nPCR) (18), and the titer was 10⁴ genome equivalents (GE) per milliliter (mL).

Two juvenile male cynomolgus monkeys (Macaca fascicularis), weighing 2.0–2.5 kg, designated as Cy1 and Cy2, were obtained from the Beijing Xierxing Institute of Biologic Resources (Beijing, China) for the cross-species infection study. For the rabbit infection study, four 7-week-old specific-pathogen free (SPF) New Zealand white rabbits, weighing 750–1,000 g, were obtained from the Department of Laboratory Animal Science of Peking University Health Science Center. Preinoculation serum and feces specimens were collected once a week for 3 weeks, and all animals were tested for alanine aminotransferase (ALT) to establish a baseline, and were confirmed as negative for antibodies against HEV by an ELISA and negative for HEV RNA by RT-nPCR. The animal experiments were approved by the Committee of Laboratory Animal Welfare and Ethics, Peking University Health Science Center. The regulations of the review committee of Laboratory Animal Welfare and Ethics and the protocol for the review on Laboratory Animal Welfare and Ethics, Peking University Health Science Center, were followed.

To determine whether rabbit HEV strains are transmissible to nonhuman primates, we inoculated intravenously 2 cynomolgus monkeys, housed separately, with 2 mL of the rabbit HEV inoculum. After inoculation, serial serum and fecal samples were collected 2×/week for 16 weeks.

Serum samples were tested for ALT levels and for IgM and IgG against HEV. All samples were also assayed for HEV RNA by RT-nPCR (15).

To clarify the extrahepatic replication sites of HEV, rabbits were experimentally infected with rabbit HEV as described (19). In brief, 4 SPF rabbits, which were housed in separate cages, were divided randomly into 2 groups (2 rabbits per group) and inoculated intravenously with either 1 mL of PBS (negative control) or 1 mL of rabbit HEV inoculum. Serum and fecal specimens were collected weekly after inoculation. Serum samples were tested for ALT activity and HEV RNA. Fecal specimens were also assayed for HEV RNA. If serum and fecal specimens became simultaneously positive for HEV RNA, a complete necropsy was performed of each rabbit. Bile and various different types of tissues and organs, including liver, kidney, small intestine, spleen, stomach, heart, brain, bladder, and lung, were collected and stored at −80°C. To prevent cross-contamination during necropsy, we used individually wrapped, sterile disposable materials and new sterile scalpel blades for each sample.

Approximately 100 mg of each tissue and organ was homogenized in 1 mL of sterile PBS (pH 7.4) to make 10% (wt/vol) suspensions and clarified by centrifugation at 4,500 g for 10 min at 4°C. Thereafter, 100 µL of the clarified supernatants was used for total viral RNA extraction, and positive-stranded and negative-stranded HEV RNA were detected by RT-nPCR as described below.

All serum samples were tested immediately for ALT levels with a Hitachi Automatic Clinical Analyzer 7180 (Hitachi High-Technologies, Tokyo, Japan), by using chemical reagents purchased from Roche (Basel, Switzerland), according to the manufacturer’s instructions. Biochemical evidence of hepatitis was recorded when the serum ALT level exceeded the baseline ALT level by >2-fold, as defined by a peak ALT value that was equal to or greater than double the prechallenge values (19,20).

The serum specimens collected from monkeys were tested for IgM and IgG against HEV by using an ELISA based on the virus E2 protein (amino acids 394–606 of HEV open reading frame [ORF] 2) (20), according to the manufacturer’s instructions (Wantai, Beijing, China). The serum samples collected from rabbits were also examined for antibodies by using the same assay. Signal-to-cutoff values were calculated, and values >1 were considered positive. Preinoculation baseline serum specimens were used as negative controls for each monkey.

RNA was extracted from 100 μL of serum, bile, tissue suspension, or 10% fecal suspension by using TRIzol reagent (Invitrogen, Burlington, ON, Canada), and purified RNA was resuspended in 11 μL of RNase-free water. To detect positive-stranded HEV RNA, 11 μL of purified RNA was reverse transcribed at 42°C for 60 min with SuperScript II reverse transcription (Invitrogen) and the external reverse primer P4 or S4 in a reaction mixture of 20 μL. Then, nested PCRs were carried out to amplify the partial fragments of ORF1 (129–373 nt) and ORF2 (5,983–6,349 nt) of the HEV genome by using the 2 sets of specific external and internal primer pairs listed in Technical Appendix Table 1). The PCR parameters for both sets of primers and both rounds of PCR were the same, with an initial incubation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 40 s, with a final incubation at 72°C for 10 min.

Tissues with detectable positive-stranded HEV RNA were then assayed for negative-sense HEV RNA by RT-nPCR with the same 2 sets of universal primers (Technical Appendix Table 1). The extracted RNA was subjected to cDNA synthesis with the external forward primer P1 or S1. Then parental RNAs were degraded by RNaseH, and this was followed by nested PCR. The amplification conditions for negative-stranded HEV RNA detection were essentially the same as those used in the detection of positive-sense HEV RNA.

The PCR protocol used in this study could detect as few as 10 GE copies of HEV plasmid DNA. Negative and positive controls were included in each assay to exclude the possibility of contamination and failure of amplification. A recombinant plasmid containing HEV ORF1 and ORF2 fragments at a concentration of 10² copies per mL and serum or fecal specimens or tissues from naive rabbits were used as positive and negative controls, respectively. Samples showing a band of the expected size on a 1.5% (w/v) agarose gel were considered positive, and the positive products were directly sequenced.

To compare the complete genome sequence of the HEV passed in the macaques to that of the inoculum, the fecal sample (rHEV-Cy1) of 1 monkey at 3 weeks’ postinoculation (wpi) and the inoculum (CHN-BJ-R14) were sequenced to determine the full-length genome as reported (21). Briefly, total RNA was extracted from 120 μL of the rabbit HEV inoculum and a 10% monkey fecal suspension in PBS by using the Total RNA Isolation System (Promega, Madison, WI, USA). cDNA was synthesized from 12 μL of purified RNA by using 1 μL (200 U) of Moloney murine leukemia virus reverse transcription (Promega) and 2 μL (10 pmol/L) of OligodT primer. With 6 sets of specific external and internal primer pairs (Technical Appendix Table 2), a set of nested PCRs were performed by using the first-strand cDNA to amplify the entire viral genome. The nested PCR was done as described (21). The nucleotide sequences at the 5′ and 3′ termini of the genome were determined by using a rapid amplification of cDNA ends (RACE) kit (Invitrogen), according to the manufacturer’s instructions.

The expected PCR products amplified from the inoculum and monkey fecal sample at 3 weeks wpi were purified and ligated into a pGEM-T vector (Promega). At least 3 positive clones for each region of the viral genome were sequenced commercially in both directions by using an automated DNA sequencer (ABI model 3730 sequencer; Applied Biosystems, Foster City, CA, USA).

Nucleotide sequences were assembled and analyzed with the MEGA 4.0 and ALIGNX software (Vector NTI package version 9.0; Invitrogen). ORFs were identified by using the EMBOSS software (version 5.0.0; emboss.sourceforge.net). The full-length genomic sequences of CHN-BJ-R14 and rHEV-Cy1 reported in this study have been deposited in GenBank under accession nos. JX109834 and JX121233, respectively.

In both of the macaques inoculated with rabbit HEV, hepatitis developed, as determined on the basis of ALT elevation, viremia, fecal shedding of viruses, and seroconversion (Figure). Dramatic elevations in serum ALT were observed 5 and 10 wpi for both monkeys, with a peak value of 135 U/L at 9 wpi for monkey Cy1 and 97 U/L at 5.5 wpi for monkey Cy2.

Before inoculation, both monkeys were seronegative for HEV and became seropositive for antibodies against HEV at 6–7 wpi. IgM against HEV was detectable from 7 to 12 wpi for Cy1 and from 6 to 8 wpi for Cy2. The rise in IgM against HEV was followed closely by a strong response of IgG against HEV for Cy1, whereas both responses occurred at about the same time for Cy2. The IgG level against HEV remained markedly elevated at the end of the 16-week experiment.

Serum and fecal samples taken before inoculation from both monkeys were negative for HEV RNA. Viremia and fecal shedding of viruses were detected in both monkeys after intravenous inoculation. Fecal excretion of rabbit HEV, indicative of replication, was first detected at 1 wpi and persisted for 5–9 weeks. HEV viremia was first detected at 5.5 wpi for Cy1 and at 2 wpi for Cy2 and lasted for 2.5–3.5 weeks. The partial sequences of the PCR products from both monkeys shared 99%–100% nucleotide identity with the original inoculum.

To analyze mutations in the rabbit HEV genome that appeared during a single passage between the 2 different host species, we sequenced rabbit HEV strains recovered from the inoculum (CHN-BJ-R14) and from experimentally infected cynomolgus monkeys (rHEV-Cy1) over the entire genome. The CHN-BJ-R14 and rHEV-Cy1 isolates had the same genomic length of 7,284 nt, excluding the 3′ poly (A) tail, and contained 3 ORFs—ORF1, ORF2, and ORF3—which encoded proteins of 1,722 aa (nt 26–5194), 660 aa (nt 5232–7214), and 113 aa (nt 5221–5562), respectively. The 5′ untranslated region (UTR) and 3′ UTR comprise 25 nt and 71 nt, respectively. Sequence analyses showed that CHN-BJ-R14 and rHEV-Cy1 shared 99.8% nucleotide identity with each other. Comparison of the complete genome sequence of rabbit HEV passed in the macaques (rHEV-Cy1) with that of the inoculum (CHN-BJ-R14) revealed 18 nt mutations over the entire genome, resulting in 9 nonsynonymous amino acid changes. ORF1 harbored 16 of the 18 nt mutations; 11 were in the helicase domain and in the RNA-dependent RNA polymerase domain (Table).

Nucleotide BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) analysis showed that CHN-BJ-R14 and rHEV-Cy1 were most closely related to genotype 3 HEV with a maximum nucleotide identity of 81%, with the exception of 3 other rabbit HEV strains isolated in Gansu (13) and Beijing (21). However, several unique features possessed only by rabbit HEVs, but not genotype 3 or other HEV strains, were observed in the 2 rabbit HEV isolates of this study. These features, discovered in a previous study (21), were characterized by an insertion of 31 aa in ORF1 (929–959 aa) and a unique A residue at nt 13 (sites based on CHN-BJ-R14) in the 5′ UTR (data not shown).

Both control rabbits remained negative for HEV RNA throughout the study. Viremia and fecal shedding of HEV were detected in rabbits inoculated with the rabbit HEV inoculum. Both rabbits were necropsied, at 5.5 wpi and 12 wpi, respectively, when ALT elevation was observed, and HEV RNA was detected simultaneously in serum and feces. Bile and 9 different types of tissues and organs were collected and tested for positive-stranded HEV RNA. Positive-stranded HEV RNA was detected in bile and in 5 of the tissues—liver, kidney, small intestine, spleen, and stomach. Detection of positive-stranded HEV RNA from various tissues and organs did not indicate that the virus was replicating in these tissues because contamination of the tissue samples by virus circulating in the blood could not be ruled out. To further identify the replicating sites of HEV, we screened for negative-stranded RNA, which is an intermediate product during HEV replication, in all tissues that were positive for the positive-stranded HEV RNA. Negative-stranded RNA was also detectable in the 5 types of tissues. The positive products were sequenced and found to be identical to the original inoculum.