Conceived and designed the experiments: HM YW. Performed the experiments: HM LZ YL CZ YM SS. Analyzed the data: HM TJH JZ YW. Contributed reagents/materials/analysis tools: YL CZ SS. Wrote the paper: HM TJH YW.
The authors have declared that no competing interests exist.
A recent study provided evidence that farmed rabbits in China harbor a novel hepatitis E virus (HEV) genotype. Although the rabbit HEV isolate had 77–79% nucleotide identity to the mammalian HEV genotypes 1 to 4, their genomic organization is very similar. Since rabbits are used widely experimentally, including as models of infection, we investigated whether they constitute an appropriate animal model for human HEV infection.
Forty-two SPF rabbits were divided randomly into eleven groups and inoculated with six different isolates of rabbit HEV, two different doses of a second-passage rabbit HEV, and with genotype 1 and 4 HEV. Sera and feces were collected weekly after inoculation. HEV antigen, RNA, antibody and alanine aminotransferase in sera and HEV RNA in feces were detected. The liver samples were collected during necropsy subject to histopathological examination.
Rabbits inoculated with rabbit HEV became infected with HEV, with viremia, fecal virus shedding and high serum levels of viral antigens, and developed hepatitis, with elevation of the liver enzyme, ALT. The severity of disease corresponded to the infectious dose (genome equivalents), with the most severe hepatic disease caused by strain GDC54-18. However, only two of nine rabbits infected with HEV genotype 4, and none infected with genotype 1, developed hepatitis although six of nine rabbits inoculated with the genotype 1 HEV and in all rabbits inoculated with the genotype 4 HEV seroconverted to be positive for anti-HEV IgG antibody by 14 weeks post-inoculation.
These data indicate that rabbits are an appropriate model for rabbit HEV infection but are not likely to be useful for the study of human HEV. The rabbit HEV infection of rabbits may provide an appropriate parallel animal model to study HEV pathogenesis.
Hepatitis E virus (HEV) is transmitted between humans by the fecal-oral route and causes an acute, self-limiting hepatitis with high morbidity in young adults. Hepatitis E is an important public health concern in many developing countries in Asia and Africa and occurs sporadically in some industrialized countries. HEV is a small, non-enveloped virus with a single-stranded, positive-sense RNA genome of approximately 7.2 kb containing three open reading frames (ORFs), ORF1, ORF2 and ORF3, where ORF3 partially overlaps ORF2
Since the first non-human strain of HEV was isolated from a pig in the United States in 1997 and designated swine HEV
As the presumed natural reservoir of HEV genotypes 3 and 4, pigs are able to amplify the virus but only develop subclinical disease
At present, the advancement of HEV research has been hampered to by the lack of an effective
The animal experimentation was approved by the Committee of Laboratory Animal Welfare and Ethics, National Institute for the Control of Pharmaceutical and Biological Products. The regulation for the review committee of laboratory animal welfare and ethics and protocol for the review on laboratory animal welfare and ethics, National Institute for the Control of Pharmaceutical and Biological Products, were followed.
The rabbit strain of HEV was originally recovered from serum samples from rabbits bred in two farms in Gansu province, China
Two strains of HEV isolated from humans were used for challenge. A genotype 4 strain (RH4) was collected from the feces of an individual with acute hepatitis E in Beijing, China and a genotype 1 strain (RH1) was collected from the feces of patients with sporadic hepatitis E in the Xinjiang Autonomous Region, China
Forty-two 7-week old SPF rabbits weighing between 800 and 1000 g were obtained from the National Institute for the Control of Pharmaceutical and Biological Products, in Beijing, China. Prior to inoculation, all rabbits were confirmed negative for anti-rabbit HEV antibodies and antigens by an enzyme-linked immunosorbent assay (ELISA).
Rabbits were divided randomly into eleven groups and inoculated intravenously with different doses of the various HEV isolates (
Inoculum strain (Groups) | Animals per group | Infectious dose: genome equivalents |
RH1 | 9 | 1.10×106 |
RH4 | 9 | 1.14×107 |
GDC9 | 2 | 1.03×103 |
RC12 | 2 | 4.69×101 |
GDC22 | 2 | 6.35×103 |
GDC37 | 2 | 2.59×104 |
GDC46 | 2 | 6.40×104 |
GDC54 | 2 | 6.93×104 |
Control | 4 | 0 |
1GDC54-18 | 4 | 6.74×106 |
2GDC54-18 | 4 | 1.35×107 |
Blood samples and feces were collected weekly following virus challenge. Serum samples were tested for levels of the liver enzyme, alanine aminotransferase (ALT), HEV antigen and anti-HEV antibodies using standard methods, as described below. Serum and feces samples were tested weekly for HEV RNA by real-time PCR, as reported previously
No gross pathological lesions were observed in the livers during necropsies. Liver samples collected at necropsy were fixed in 10% neutral buffered formalin and processed for routine histological examination. Each specimen was embedded in paraffin and cut into 3 µm serial sections, stained with hematoxylin–eosin and subjected to histopathological examination by light microscopy.
All rabbits were monitored weekly after inoculation for 14 weeks. ALT concentrations in sera were measured on the day of collection using an automated analyzer (Hitachi 912; Roche, Indianapolis, USA) according to the manufacturer's instructions. Biochemical evidence of hepatitis was recorded when the serum ALT concentration exceeded the baseline ALT level by more than two-fold, as defined by a peak ALT value that was equal to or greater than twice the pre-challenge values. These criteria were based on the correlation between the level of ALT increase and liver pathology described in a previous study using a non-human primate as an animal model of hepatitis
All serum samples were tested for the presence of HEV antigen as described previously
To determine the genomic dose of each inoculum, RNA was extracted from sera and fecal suspensions using a spin-column kit and the procedures of Kinghawk Biopharmaceutical Company (Beijing, China) and evaluated by a real-time fluorescence RT-PCR assay with primers based on the conserved regions of HEV ORF3, as reported previously
Clinical signs, such as diarrhea and jaundice, were not observed; however, some rabbits decreased food consumption for part of the duration of the study. Some rabbits suffered accidental death during blood collection by heart puncture.
Prior to inoculation, all rabbits were seronegative for HEV. All control rabbits remained seronegative throughout the study. Anti-HEV IgG was detected in six of nine rabbits inoculated with the RH1 strain HEV (group RH1) and in eight of nine rabbits inoculated with the RH4 strain (group RH4) by 14 weeks post-inoculation (wpi) (
(A) The mean ELISA signal-to-cutoff (S/CO) values for all rabbits from each group at each week post-inoculation are plotted. (B) All rabbits from groups 3 to 8 inoculated with the non-passaged HEV are named R1ST and the combination of groups RH1 and RH4 are named RH.
Inoculum strain (Groups) | No. of seropositive rabbits/total no. tested at indicated weeks post-inoculation | ||||||||||||||
0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | |
RH1 | 0/9 | 0/9 | 0/9 | 0/9 | 0/9 | 0/9 | 1/9 | 2/9 | 5/9 | 5/9 | 5/9 | 6/9 | 6/9 | 6/9 | 6/9 |
RH4 | 0/9 | 0/9 | 0/9 | 0/9 | 0/9 | 1/9 | 2/9 | 4/9 | 4/8 | 4/8 | 4/8 | 4/8 | 7/8 | 7/8 | 7/8 |
GDC9 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 1/2 | 1/2 | 0/2 | 0/2 | 1/1 | 1/1 | 1/1 | 1/1 | 1/1 | 1/1 |
RC12 | 0/2 | 0/2 | 0/2 | 0/2 | 1/2 | 2/2 | 2/2 | 2/2 | 2/2 | 2/2 | 2/2 | 2/2 | 2/2 | 2/2 | 2/2 |
GDC22 | 0/2 | 0/2 | 0/2 | 0/2 | 1/2 | 1/2 | 1/2 | 1/2 | 1/2 | 2/2 | 2/2 | 2/2 | 2/2 | 2/2 | 2/2 |
GDC37 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 1/2 | 1/2 | 1/2 | 1/2 | 1/2 | 2/2 | 2/2 | 2/2 |
GDC46 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 1/2 | 1/2 | 1/2 | 1/2 | 1/2 | 2/2 | 2/2 | 2/2 | 2/2 |
GDC54 | 0/2 | 0/2 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 1/1 | 1/1 | 1/1 | 0/1 | 0/1 | 0/1 |
Control | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 |
1GDC54-18 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/3 | 1/3 | 1/3 | 2/3 | 1/3 | 2/3 | 2/3 | 1/1 | 1/1 | 1/1 |
2GDC54-18 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 1/4 | 1/4 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 |
All rabbits were negative for HEV antigen prior to inoculation and no control rabbits seroconverted throughout the study. The mean S/CO values of antigenemia differed between the rabbits inoculated with genotypes 1 and 4 and rabbits inoculated with the non-passaged rabbit HEV isolates (p<0.0001) (
(A) The weekly mean ELISA signal-to-cutoff (S/CO) values for all rabbits from each group are plotted. (B) All rabbits from groups 3 to 8 inoculated with the non-passaged HEV are named R1ST and the combination of groups RH1 and RH4 are named RH.
Inoculum (Groups) | No. of seropositive rabbits/total no. tested at indicated weeks post-inoculation | ||||||||||||||
0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | |
RH1 | 0/9 | 0/9 | 0/9 | 0/9 | 0/9 | 0/9 | 0/9 | 0/9 | 0/9 | 0/9 | 0/9 | 0/9 | 0/9 | 0/9 | 0/9 |
RH4 | 0/9 | 0/9 | 1/9 | 1/9 | 1/9 | 0/9 | 1/9 | 1/9 | 1/8 | 1/8 | 1/8 | 1/8 | 0/8 | 1/8 | 1/8 |
GDC9 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 1/2 | 1/2 | 1/2 | 0/2 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 |
RC12 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 |
GDC22 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 |
GDC37 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 1/2 | 1/2 | 1/2 | 1/2 | 1/2 | 1/2 | 1/2 | 1/2 | 1/2 | 1/2 |
GDC46 | 0/2 | 1/2 | 1/2 | 1/2 | 1/2 | 1/2 | 1/2 | 1/2 | 1/2 | 1/2 | 1/2 | 0/2 | 0/2 | 0/2 | 0/2 |
GDC54 | 0/2 | 2/2 | 1/1 | 1/1 | 1/1 | 1/1 | 1/1 | 1/1 | 1/1 | 1/1 | 1/1 | 1/1 | 1/1 | 1/1 | 1/1 |
Control | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 |
1GDC54-18 | 0/4 | 0/4 | 1/4 | 1/4 | 1/4 | 1/3 | 1/3 | 1/3 | 1/3 | 2/3 | 1/3 | 1/3 | 0/1 | 0/1 | 0/1 |
2GDC54-18 | 0/4 | 0/4 | 0/4 | 3/4 | 3/4 | 2/4 | 2/4 | 2/4 | 2/4 | 2/4 | 2/4 | 2/4 | 2/4 | 2/4 | 2/4 |
Pre-inoculation samples taken from all rabbits were negative for HEV RNA and all uninoculated control rabbits remained negative throughout the experiment. Rabbit HEV RNA was detected variably in serum and fecal samples from rabbits in the various groups (
Inoculum (Groups) | No. of positive sera (no. of positive feces)/total no. tested at indicated weeks post-inoculation | ||||||||||||||
0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | |
RH1 | 0(0)/9 | 0(0)/9 | 0(0)/9 | 0(0)/9 | 0(0)/9 | 0(0)/9 | 0(0)/9 | 0(0)/9 | 0(0)/9 | 0(0)/9 | 0(0)/9 | 0(0)/9 | 0(0)/9 | 0(0)/9 | 0(0)/9 |
RH4 | 0(0)/9 | 2(0)/9 | 2(2)/9 | 2(1)/9 | 1(1)/9 | 0(0)/9 | 1(1)/9 | 0(1)/9 | 1(1)/8 | 0(0)/8 | 1(0)/8 | 1(1)/8 | 0(1)/8 | 1(0)/8 | 0(0)/8 |
GDC9 | 0(0)/2 | 0(0)/2 | 0(1)/2 | 0(0)/2 | 0(1)/2 | 0(2)/2 | 0(1)/2 | 0(1)/2 | 1(1)/2 | 0(1)/1 | 0(0)/1 | 0(0)/1 | 0(0)/1 | 0(0)/1 | 0(0)/1 |
RC12 | 0(0)/2 | 0(0)/2 | 0(0)/2 | 0(0)/2 | 0(0)/2 | 0(0)/2 | 0(0)/2 | 0(0)/2 | 0(0)/2 | 0(0)/2 | 1(0)/2 | 0(0)/2 | 0(0)/2 | 0(0)/2 | 0(0)/2 |
GDC22 | 0(0)/2 | 0(0)/2 | 0(0)/2 | 0(0)/2 | 0(0)/2 | 0(1)/2 | 0(0)/2 | 0(1)/2 | 0(1)/2 | 0(1)/2 | 0(1)/2 | 0(1)/2 | 0(1)/2 | 0(1)/2 | 0(1)/2 |
GDC37 | 0(0)/2 | 0(0)/2 | 0(0)/2 | 0(2)/2 | 0(2)/2 | 0(1)/2 | 0(1)/2 | 0(1)/2 | 0(1)/2 | 0(1)/2 | 0(1)/2 | 0(1)/2 | 0(1)/2 | 0(1)/2 | 0(1)/2 |
GDC46 | 0(0)/2 | 0(0)/2 | 0(0)/2 | 0(0)/2 | 0(1)/2 | 0(1)/2 | 0(1)/2 | 0(1)/2 | 0(1)/2 | 0(1)/2 | 1(2)/2 | 0(0)/2 | 0(0)/2 | 0(0)/2 | 0(0)/2 |
GDC54 | 0(0)/2 | 0(0)/2 | 0(0)/1 | 0(1)/1 | 0(1)/1 | 0(1)/1 | 0(1)/1 | 0(1)/1 | 0(1)/1 | 0(1)/1 | 0(1)/1 | 0(1)/1 | 0(1)/1 | 0(1)/1 | 0(1)/1 |
Control | 0(0)/4 | 0(0)/4 | 0(0)/4 | 0(0)/4 | 0(0)/4 | 0(0)/4 | 0(0)/4 | 0(0)/4 | 0(0)/4 | 0(0)/4 | 0(0)/4 | 0(0)/4 | 0(0)/4 | 0(0)/4 | 0(0)/4 |
1GDC54-18 | 0(0)/4 | 0(1)/4 | 1(0)/4 | 2(0)/4 | 0(1)/4 | 1(1)/3 | 1(2)/3 | 0(1)/3 | 0(1)/3 | 0(1)/3 | 0(1)/3 | 0(1)/3 | 0(0)/1 | 0(0)/1 | 0(0)/1 |
2GDC54-18 | 0(0)/4 | 2(1)/4 | 1(0)/4 | 2(2)/4 | 2(3)/4 | 0(1)/4 | 1(3)/4 | 2(3)/4 | 2(3)/4 | 2(1)/4 | 1(1)/4 | 1(1)/4 | 0(1)/4 | 0(1)/4 | 1(2)/4 |
Viremia was detected in sera from 1 to 3 wpi in two of nine RH4 group rabbits inoculated with genotype 4 HEV, with one animal intermittently positive thereafter. No viral RNA was detected in any rabbit of the RH1 group inoculated with HEV genotype 1. Viremia was detected only three times among any of the six groups of rabbits inoculated with the non-passaged rabbit HEV isolates, including one of two rabbits in the GDC9 group at 8 wpi, one of two in the RC12 group at 10 wpi and one of two in the GDC46 group at 10 wpi. For the 1GDC54-18 group rabbits, viremia was detected from 1 to 2 wpi and 5 to 6 wpi. HEV RNA was first detected in the serum of two of four rabbits in the 2GDC54-18 group at 1 wpi and was intermittently positive thereafter, up to 11 wpi.
Fecal shedding of viruses was not detected in group RH1. For group RH4 rabbits, HEV RNA was first detected in feces from two of the nine rabbits at 2 wpi and one them was intermittently positive thereafter. All of the groups inoculated with the non-passaged rabbit HEV isolates were intermittently positive for HEV RNA in feces, except group RC12. For the group 1GDC54-18 rabbits, HEV RNA was shed in the feces first at 1 wpi and lasted until 4 to 11 wpi. Three of the four group 2GDC54-18 rabbits were HEV RNA positive at 4 wpi and from 6 to 8 wpi.
The viruses recovered from selected experimentally-infected rabbits were sequenced to confirm that they were derived from the original inoculum, with nucleotide identity 99–100% to the original inoculum.
No instant elevations in serum ALT-levels were observed in the four control rabbits, nine rabbits each in groups RH1 and RH4 during the entire study (
(A) The mean ALT values of all rabbits from each group at each week post-inoculation are plotted. (B) All rabbits from groups 3 to 8 inoculated with the non-passaged rabbit HEV are named R1ST and the combination of groups RH1 and RH4 are named RH.
No statistical data on the pathological signs of HEV infection in the liver can be reported because liver biopsies were not performed and no rabbits were regularly necropsied during the entire study. Some rabbits that showed elevated ALT levels when they were necropsied at the end of the study or died due accidentally and all control rabbits were investigated for liver pathology. Multifocal lymphohistiocytic infiltrates were distributed irregularly in the liver of a rabbit from group RH4 (
(A & B) Liver sections from a group 2GDC54-18 rabbit showing localized extensive hepatocellular necrosis (magnification 10× and 20×, respectively). (C) Liver section from a group RH4 rabbit showing irregularly distributed multifocal lymphohistiocytic infiltrates (20×). (D) Liver section from a control rabbit showing no visible pathological signs of HEV infection (20×).
The lack of an effective cell culture system and a small animal model has hampered the progress of HEV research. Experimental infections of pigs and non-human primates with HEV have been well documented
Six original isolates of rabbit HEV were used to infect 12 SPF rabbits. Based on a 304 bp region of ORF2, the rabbit HEV strains used in this study shared 84–99% identity and are 73–77, 70–76, 75–82, 71–77, and 53–65% identical to the corresponding regions of human HEV genotypes 1, 2, 3, 4, and avian HEV, respectively
Because the virulence of the first-generation rabbit HEV was limited, we used virus recovered from the feces of rabbits inoculated with the GDC54 strain of HEV as a second passage inoculum for the further studies. The second passage HEV GDC54-18 virus inoculum was used to infect another eight rabbits, using two different doses. As expected, challenging with higher doses of virus in terms of genome equivalents caused a more severe hepatitis. Serum ALT levels, which are indicative of recent liver damage and suggestive of an acute infection, peaked at 10 wpi (
Another objective of this study was to determine whether human HEV could infect rabbits. So far, no evidence has proved that human genotypes 1 and 2 HEV can infect pigs, although genotypes 3 and 4 can infect both human and pigs. In addition to pigs, many other animals have been shown to be susceptible to infection with genotypes 3 and 4 HEV and can serve as a reservoir of HEV that can amplify the virus and transmit to humans
In summary, we infected rabbits successfully with rabbit HEV and the clinical picture in these animals was similar to HEV infection of monkeys. It seems that rabbits constitute a valuable model to study the mechanisms of HEV replication and pathogenesis. These studies indicate the rabbit model has potential for the development of HEV vaccines and clinical therapies for the treatment of infection.
We thank Zhi Lin and Yanwei Yang for assistance with pathological testing.