Effect of swine hepatitis E virus on the livers of experimentally infected Mongolian gerbils by swine hepatitis E virus.

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Contents lists available at ScienceDirect

Virus Research journal homepage: www.elsevier.com/locate/virusres

Effect of swine hepatitis E virus on the livers of experimentally infected Mongolian gerbils by swine hepatitis E virus

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Yifei Yang a,b , Ruihan Shi a , Ruiping She a,∗ , Majid Hussain Soomro a , Jingjing Mao a,c , Fang Du a , Yue Zhao a , Can Liu a a Lab of Animal Pathology and Public Health, College of Veterinary Medicine, China Agricultural University; Key Laboratory of Zoonosis of Ministry of Agriculture, China Agricultural University, Beijing 100193, China b Institute of Chinese Materia Medica, China Academy of Chinese Medical Science, Beijing 100700, China c National Shanghai Center for New Drug Safety Evaluation Research Center, Shanghai 201203, China

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a r t i c l e

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a b s t r a c t

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Article history: Received 22 March 2015 Received in revised form 3 June 2015 Accepted 5 June 2015 Available online xxx

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Keywords: Hepatitis E virus Mongolian gerbil Liver Lesion

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1. Introduction

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Previous studies have shown that hepatitis E virus (HEV) can be transmitted between rats, pigs, cattle, rabbits, chicken, cats, and deer. Because wild and domestic rodents have anti-HEV antibodies, they are considered potential reservoirs of HEV. In the current study, Mongolian gerbils were experimentally infected with swine hepatitis E virus and the effects of this infection were investigated. After inoculation with HEV, the liver-to-body weight ratio increased at 7 dpi. Mongolian gerbils demonstrated significant increase (p < 0.05) in Aspartate Transaminase (AST), alanine transaminase (ALT) and total bilirubin (TBIL) concentrations in the sera, and HEV IgG was detected at 21 days post-inoculation (dpi). Real-time PCR revealed that the copies of HEV RNA in the liver were detected at 7 dpi, and peaked at 28 dpi at a concentration of 7.73 logs g−1 . Using both light and electron microscopy, hepatic lesions were observed in the HEV inoculated animals. In the experimental group, characteristic viral hepatitis lesions were prominent in the liver. HEV antigen was detected in the liver by immunohistochemistry, and HEV ORF3 antigen was detectable in liver by Western blot. These results clearly demonstrate that viral load of HEV in livers was dynamic, and ultrastructural hepatic injury in HEV infected Mongolian gerbils and anti-HEV IgG positive seroconversion were observed during infection. © 2015 Published by Elsevier B.V.

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Hepatitis E virus (HEV) can cause hepatitis in humans and many other species. HEV is a non-enveloped, positive sense singlestranded RNA virus that belongs to the family Hepeviridae (Smith et al., 2014). HEV has a 7.2 kb RNA genome (Emerson and Purcell, 2003; Tam et al., 1991) that contains three open reading frames (ORF), ORF1, ORF2 and ORF3 (Panda et al., 2007). HEV isolated from human, pig, wild boar, deer, mongoose, rabbit, and camel belongs to

Abbreviations: HEV, hepatitis E virus; HE, hepatitis E; dpi, day post-inoculation; PCR, polymerase chain reaction. ∗ Corresponding author at: Lab of Animal Pathology and Public Health, Key Laboratory of Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. Tel.: +86 10 62733060; fax: +86 10 62733321. E-mail addresses: [email protected] (Y. Yang), [email protected] (R. Shi), [email protected] (R. She), [email protected] (M.H. Soomro), [email protected] (J. Mao), [email protected] (F. Du), [email protected] (Y. Zhao), [email protected] (C. Liu).

the Orthohepevirus A taxonomy. HEV isolated from chicken belongs to Orthohepevirus B taxonomy. HEV isolated from rat belongs to Orthohepevirus C taxonomy, and HEV isolated from bat belongs to Orthohepevirus D taxonomy (Smith et al., 2014). HEV has become an important public health problem, especially in developing countries. HEV can be transmitted between rats, pigs, cattle, rabbits, chicken, cats, and deer (Feagins et al., 2008; Huang et al., 2004; Okamoto et al., 2004; Tei et al., 2004; Kabrane-Lazizi et al., 1999; Worm et al., 2002; Zhao et al., 2009). HEV is generally transmitted through a fecal–oral route (Worm et al., 2002; Hsieh et al., 1999). According to seroprevalence surveys, the numbers of unrecognized or subclinical infections are under-estimated in many countries (Ijaz et al., 2005; Xiao et al., 2012; Berto et al., 2012; Hakze-van der Honing et al., 2011). Because anti-HEV antibodies have been detected in wild and domestic rats, rodents are considered a potential reservoir of HEV (Kabrane-Lazizi et al., 1999; Huang et al., 2009; Maneerat et al., 1996). Although rodents, pigs, non-human primates, and rabbits have been previously used as animal models in hepatitis studies (Huang et al., 2009; Dandri and Lütgehetmann, 2014; Yu et al., 2014; Krawczynski et al., 2011),

http://dx.doi.org/10.1016/j.virusres.2015.06.007 0168-1702/© 2015 Published by Elsevier B.V.

Please cite this article in press as: Yang, Y., et al., Effect of swine hepatitis E virus on the livers of experimentally infected Mongolian gerbils by swine hepatitis E virus. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.06.007

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removed quickly, washed thoroughly with normal saline to remove residual blood and then weighed. Fresh frozen tissue samples were collected from the livers during the necropsy and stored at −86 ◦ C for HEV RNA detection and Western-blotting. Additional tissue from each liver was collected and fixed in neutral 4% paraformaldehyde for 3 days for histopathological and immunohistochemical analyses. Certain tissues were also fixed in 2.5% (v/v) glutaraldehyde-polyoxymethylene solution for 6–8 h in preparation for transmission electron microscopy (TEM).

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data on HEV pathogenesis are sparse, especially regarding acute infections. Additionally, an efficient cell culture system for hepatitis studies is not currently available (Krawczynski et al., 2011). Our lab has successfully infected Mongolian gerbils using an inoculation of swine HEV. HEV was consistently detected in the liver, kidney, spleen, and small intestine (Li et al., 2009). To better understand the effects of swine HEV on the livers of experimentally infected Mongolian gerbils, we evaluated the liver-to-body weight ratio, viral load, histopathological changes, antigen distribution in the liver, serum liver enzyme levels (ALT, AST and T-BIL), and dynamic changes in HEV antibody levels in Mongolian gerbils.

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2. Materials and methods

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2.1. Ethics statement

The ELISA for the detection of anti-HEV IgG was performed following the manufacturer’s instructions (Wantai, Beijing, China).

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2.6. ELISAs for HEV antibody and antigen detection

2.7. Serum biochemistry detection of liver enzymes

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All animal experiments were approved by the Animal Care and Use Committee of China Agricultural University (CAU) (permit number: 20140115-089). We followed the guidelines of the CAU Animal Care and Use Committee when handling the experimental animals during this study.

The levels of ALT, AST, and T-BIL in serum were measured using an automated biochemistry analyzer (HITICHI7160, Japan) according to the manufacturer’s instructions.

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2.2. Virus

2.8. RNA extraction The samples (100 mg of liver tissue) were disrupted with liquid nitrogen (with the exception of the serum samples). RNA was extracted using the Ultrapure RNA kit and the RNApure Virus kit (CWBIO, Beijing, China) following the manufacturer’s instructions. The RNA was stored at −86 ◦ C.

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The strain of swine HEV used, a genotype 4 virus, was derived from the second passage of a liver sample from an SPF swine infected with swine HEV (CHN-HB-HD-L2, GenBank accession number KM024042). A 10% (g/mL) homogenate of HEV-positive liver was prepared (Huang et al., 2009) and titered using realtime PCR, as previously described (Zhao et al., 2007; Jothikumar et al., 2006; Zwettler et al., 2012). The homogenate with a titer of 6.57 × 108 genome equivalents (GE) per mL was stored at −86 ◦ C.

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2.3. Animals

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71 72 73 74 75 76 77

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Eighty-four male Mongolian gerbils (Meriones unguiculatus), with body weights between 50 and 60 g and aged 8–10 weeks old, were purchased from the Department of Experimental Animal Sciences of Capital Medical University (Beijing, China). Their food and water were sterilized, and the experimental animal rooms were fumigated. To avoid stressing the Mongolian gerbils, all experiments were conducted following an acclimation period of 3 days after their arrival at the facility. Prior to inoculation with HEV, the Mongolian gerbils were confirmed negative for HEV antibodies by ELISA assay and no HEV antigen was detected in the serum or feces of experimental animals by nested-PCR. The gerbils in the experimental and control groups were quarantined in two different experimental animal rooms.

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2.4. Experimental design

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2.9. Real-time RT-PCR assays Primers and probes targeting the HEV ORF3 region and consisting of the following sequences were used: forward primer HEVORF3-S: 5 -GGTGGTTTCTGGGGTGAC-3 , reverse primer HEVORF3-AS: 5 -AGGGGTTGGTTGGATGAA-3 , and probe 5 -FamTGATTCTCAGCCCTTCGC-Tamra-3 (Jothikumar et al., 2006). Following the method described by Priscilla F. Gerber, purified high-quality plasmid DNA was prepared for absolute quantification from the HEV ORF3 region (Gerber et al., 2014). The amount of the recombinant plasmid which was synthesized by the BGI company (China) was quantified using a Scandrop RS232 spectrophotometer according to the manufacturer’s instructions (Analytikjena, Germany) and converted into genome copy numbers (Gerber et al., 2014). A standard curve was generated using plasmid DNA, and the HEV titers of the samples were determined based on the standard curve. Quantitative detection of HEV was conducted using separate reactions in triplicate. We used 1-step real-time reverse transcription PCR on the Bio-Rad iQ5 system (Bio-Rad, USA) in a 96-well format under the following conditions: 50 ◦ C for 15 min for reverse transcription, 95 ◦ C for 10 min for initial denaturation followed by 45 cycles of amplification with denaturation at 95 ◦ C for 20 s, and annealing and extension at 58 ◦ C for 40 s. The 50 ␮L reaction mixture consisted of Ultra SYBR One Step qRT-PCR buffer (CWBIO, Beijing, China) 25 ␮L, SuperEnzyme Mix 2 ␮L, 300 nmol/L of primers, and 100 nmol/L of probe and 15 ␮L of RNA. Negative and non-template controls were included to exclude non-specific amplification.

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Eighty-four gerbils were randomly divided into two groups. The experimental group and the control group each comprised 42 gerbils. Six gerbils were euthanized for necropsies at each time point in each group. Each gerbil in the experimental group was inoculated with 0.1 mL of the above-mentioned prepared viral homogenate by intraperitoneal injection. Gerbils in the control group were inoculated with an equal volume of homogenate from an SPF swine liver that tested negative for HEV by RT-PCR. All gerbils were given food and water ad libitum during the experimental period.

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2.5. Sampling

2.10. Histopathological examinations

Serum was collected at 0, 7, 14, 21, 28, 42, and 56 day postinoculation (dpi) and was stored at −86 ◦ C. Body weights of gerbils were measured before necropsy. After euthanasia, livers were

2.10.1. Light microscopy (LM) The fixed livers were processed in paraffin using a standard protocol, and 4 ␮m sections were prepared and stained with

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There was no evidence of clinical disease in the HEV experimental group.

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3.2. Liver-to-body weight ratio

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Liver-to-body weight ratio was obtained by comparing the liver weights to the body weight of euthanized gerbils. There was no obviously in difference of body weight among the gerbils from the control and inoculated groups (Table 1). However, at 7 dpi, 14 dpi, 21 dpi, 28 dpi, and 42 dpi, the weights of the livers from the inoculated group were significantly higher than those from the control

2.25 ± 0.32 64.37 ± 5.77 0.0352 ± 0.0013 2.34 ± 0.50 70.97 ± 11.37 0.0335 ± 0.0025

Control 56 dpi

Inoculated Control

Control Inoculated

2.79 ± 0.47* 80.51 ± 7.80 0.0346 ± 0.0027 2.32 ± 0.23 70.60 ± 5.37 0.0329 ± 0.0018

Control

2.20 ± 0.15 68.86 ± 3.47 0.0319 ± 0.0018 3.31 ± 0.45** 70.20 ± 7.47 0.0471 ± 0.0099** *p < 0.05; **p < 0.01. a Data are means ± SD.

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2.25 ± 0.21 71.39 ± 4.49 0.0315 ± 0.0013

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2.81 ± 0.39* 72.58 ± 8.48 0.0387 ± 0.0037**

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Liver weight (g) Body weight (g) Liver-to-body weight ratio (g/g)

3.1. No significant clinical signs after HEV inoculation

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42 dpi

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Inoculated

3. Results

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Control

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Inoculated

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A small amount of Mongolian gerbil livers from each group were homogenized in lysis buffer containing 7 M urea, 2 M thiourea, 4% Chaps, 1% DTT, 400 mM Tris-base, and 1 mM PMSF. After centrifugation at 12,000 rpm for 20 min at 4 ◦ C, the supernatant was collected and used as tissue lysate. Protein concentrations were determined by Nanodrop 2000 spectrophotometer (Thermo, USA). Equal amounts of protein from each sample were boiled in SDS sample buffer for 10 min. The samples were separated by SDS-PAGE under reducing conditions and electroblotted onto polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were then blocked by 5% skim milk for 1 h and incubated with the primary monoclonal antibody HEV ORF3 (1:300 diluted, Beijing Protein Institute, Beijing, China) overnight at 4 ◦ C. After washing, the membranes were incubated with a secondary antibody, horseradish peroxidase-conjugated goat anti-mouse IgG, at 37 ◦ C for 1 h. The conjugated substrate was detected with an enhanced chemiluminescence detection kit (CWBIO, China) and exposed to X-ray film. Equal protein loading was confirmed by staining with beta-actin antibody (Wuhan Boster Bio-Engineering Limited Company, China).

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28 dpi

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Group

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DPI

180

2.91 ± 0.38* 78.54 ± 7.36 0.0369 ± 0.0024

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Inoculated

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1.94 ± 0.19 69.98 ± 3.67 0.0277 ± 0.0016

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Control

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Inoculated

2.12. Western-blotting analysis

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2.19 ± 0.17 72.44 ± 2.93 0.0303 ± 0.0018

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Control

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Sections were prepared as previously described in this section. Monoclonal mouse anti-HEV ORF2 antibody (1:200 dilution; Beijing Protein Institute, Beijing, China) was the primary antibody used. Immunohistochemical staining was performed following the instructions that were included in the HistostainTM -Plus kit (ZSGB-BIO, Beijing, China). 3,3 -Diaminobenzidine tetrahydrochloride (DAB; ZSGB-BIO, Beijing, China) was applied for 5 min to visualize the antigen–antibody compound, after which Gill’s hematoxylin was applied as the background stain. The same procedure was conducted on the control group samples. The slides were observed under an Olympus microscope (Japan).

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2.17 ± 0.27a 71.67 ± 3.64 0.0303 ± 0.0048

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Inoculated

169

Liver weight (g) Body weight (g) Liver-to-body weight ratio (g/g)

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Group

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21 dpi

166

14 dpi

2.11. Immunohistochemical staining for HEV antigen

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7 dpi

172

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0 dpi

171

2.10.2. Transmission electron microscopy (TEM) For TEM, the liver samples were cut into pieces (2 × 2 mm) and fixed in 2.5% (v/v) glutaraldehyde-polyoxymethylene solution for 6–8 h. The samples were then washed and post fixed in 2% OsO4 for 1 h at 4 ◦ C. The tissues were dehydrated using ascending grades of ethanol and embedded in araldite CY212. Ultra-thin sections (60–70 nm) were cut and stained with alkaline and lead citrate uranyl acetate. The sections were observed under a JEM 100CX transmission electron microscope.

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2.44 ± 0.21* 68.94 ± 6.94 0.0355 ± 0.0035**

hematoxylin and eosin for histological evaluation. All tissue sections were observed under an Olympus microscope (Japan).

DPI

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Table 1 Livers weights and body weights of the experimentally infected Mongolian gerbils.

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2.23 ± 0.20 70.47 ± 5.52 0.0316 ± 0.0012

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at 6.8017 ␮mol/L, which is approximately twofold higher than the baseline level (the average baseline value was 3.93 ␮mol/L) (Fig. 2C). The levels of ALT, AST and T-BIL were normal in the control group throughout the study.

3.5. Real-time PCR detection of HEV RNA in gerbil livers

Fig. 1. Anti-HEV IgG in Mongolian gerbils as determined by ELISA. In the inoculated group, anti-HEV IgG in the serum was positive at 21 dpi to 42 dpi. No changes were seen in the control group. The cutoff value used is 0.217, which is the mean value of the negative control.

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group (p < 0.05; Table 1). The liver-to-body weight ratio of the inoculated group was significantly higher than that of the control group at 7 dpi, 28 dpi, and 42 dpi (Table 1).

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3.3. Serum anti-HEV IgG was detected by ELISA assay

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Anti-HEV IgG was first detected on day 21 dpi, and a positive signal was detected in the serum until 42 dpi (Fig. 1). The antiHEV IgG positive detection rate at each time point is also shown in Fig. 1. All assays were performed in triplicate, and the data are expressed as means (±standard deviation). The mean values of the OD450/630 nm for the two groups were analyzed using Thermo System software. The gerbils in the control group were negative for anti-HEV IgG.

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3.4. Increased levels of hepatic enzymes after HEV inoculation

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The concentrations of hepatic enzymes are shown in Table 2. The ALT levels in the experimental group were significantly higher than in the control group (p < 0.05). At 21 dpi and 28 dpi, ALT levels in the experimental group were approximately 2-fold higher than the baseline level, and ALT concentrations reached 98.37102.15 U/L. The peak ALT level measured was 102.15 U/L in the experimental group, which was at least twofold higher than the baseline value (Fig. 2A). The average ALT level in the control group was 49.25 U/L. Dramatic increases in AST levels were observed at 14 dpi and 21 dpi in the experimental group. The observed AST levels in serum peaked at 352.69 U/L, which is more than 2.3-fold higher than the baseline level of 148.97 U/L (Fig. 2B). The T-BIL levels were also notably higher after inoculation and peaked at 21 dpi

A standard curve was established using a corresponding cloned amplicon that was serially diluted from 101 to 109 copies/␮L and amplified in triplicate. The linear correlation (R2 ) between the CT and the copy number logarithm was 0.999, the slope of the standard curve was −3.380, and the intercept was 45.34. The real-time PCR results are summarized in Table 3. At 7 dpi, HEV RNA was detected in the liver (6/6). The rates of positive detection ranged from 83.3% (5/6) to 100% (6/6) during HEV infection. All the livers from gerbils in the control group were negative for HEV RNA. The HEV viral load results are shown in Table 3. HEV RNA was detected at 7 dpi with a viral load of 5.15 logs g−1 , after which it increased and peaked at 7.73 logs g−1 at 28 dpi. At 42 dpi, the HEV concentration in the liver decreased to 4.52 logs g−1 and was not detectable at 56 dpi. All livers from gerbils in the control group were negative for HEV RNA.

3.6. Liver histopathology with light microscopy

3.7. Ultrastructural analysis of the liver using TEM

Table 2 Concentrations of hepatic enzymes in experimentally infected Mongolian gerbils. DPI

0 dpi

Group

Inoculated

Control

Inoculated

Control

Inoculated

Control

Inoculated

Control

ALT (U/L) AST (U/L) T-BIL (␮mol/L)

45.64 ± 3.48a 120.46 ± 4.17 3.66 ± 0.13

48.27 ± 8.98 116.03 ± 4.92 3.84 ± 0.13

79.35 ± 7.19** 177.83 ± 5.42** 4.26 ± 0.55*

48.11 ± 6.09 133.28 ± 5.16 3.58 ± 0.52

83.85 ± 10.65** 225.13 ± 5.44** 4.97 ± 0.34**

48.69 ± 6.39 125.68 ± 5.41 3.91 ± 0.13

98.37 ± 7.45** 267.29 ± 8.40** 6.80 ± 0.22**

50.51 ± 4.11 125.52 ± 6.13 3.92 ± 0.13

21 dpi

DPI

28 dpi

Group

Inoculated

Control

Inoculated

Control

Inoculated

Control

ALT (U/L) AST (U/L) T-BIL (␮mol/L)

102.15 ± 7.96** 340.32 ± 7.52** 6.01 ± 0.29**

47.20 ± 6.02 127.70 ± 5.11 3.86 ± 0.11

90.42 ± 9.38** 352.69 ± 11.84** 4.85 ± 0.17**

53.03 ± 8.55 126.97 ± 5.54 3.87 ± 0.12

91.73 ± 9.40** 278.77 ± 6.40** 4.77 ± 0.33*

47.98 ± 4.42 121.91 ± 3.69 4.12 ± 0.16

42 dpi

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In the control group, no apparent liver damage was observed. The electron density in the cell plasma was high (Fig. 4A). Spherical and elongated mitochondria were abundant, and the mitochondria cristae and the endoplasmic reticulum cisternae were smooth (Fig. 4B and C). In the experimental group, the electron density in the cell plasma was lower (Fig. 4D), and the mitochondria were swollen and had thin cristae (Fig. 4E and G). The cytoskeleton was severely disassembled, and diffuse, petal-like particles were observed in the cytoplasm (Fig. 4F).

14 dpi

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Histopathological examination of livers from the experimental group revealed multifocal, mild hepatitis characterized by the infiltration of a large number of lymphocytes and plasma cells in the portal tracts and hepatic parenchyma. Varying levels of inflammatory cell infiltration were observed at different time points, as shown in Fig. 3. The number of inflammatory cells in the portal tracts peaked at 28 dpi (Fig. 3E). No gross histopathological lesions were observed in the liver sections from the control group (Fig. 3A).

7 dpi

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56 dpi

*p < 0.05; **p < 0.01. a Data are means ± SD.


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Fig. 2. Changes in enzymatic activities of ALT, AST and T-BIL. (A) The level of ALT was significantly increased in the experimental group compared with the control group (p < 0.05). At 21 dpi and 28 dpi, levels of ALT were elevated by approximately twofold higher above baseline levels. The peak level of ALT was 102.15 U/L in the experimental group, which is at least two folds higher than baseline values. (B) AST was significantly elevated from 14 dpi to 21 dpi (p < 0.05). The level of AST in serum peaked at 352.69 U/L at 42 dpi, more than 2.3-fold above the baseline value of 148.97 U/L. (C) Levels of T-BIL were elevated following inoculation (p < 0.05). At 21 dpi, concentrations of T-BIL were significantly increased compared to 14 dpi and peaked at 6.8017 ␮mol/L, which is approximately twofold above baseline levels (the average baseline value was 3.93 ␮mol/L). (*, p < 0.05; **, p < 0.01).

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3.8. Immunohistochemical staining for HEV antigen

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Using immunohistochemical staining, HEV antigen was detected in the liver of all the gerbils in the experimental group between 7 dpi and 56 dpi. Positive signals were observed in the cytoplasm of hepatocytes (Fig. 5B). No positive HEV antigen signal was detected in any tissue from gerbils in the control group (Fig. 5A).

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3.9. Western blotting for HEV ORF3 antigen

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According to the results of Western-blotting, HEV ORF3 antigen was detected in the liver during infection (Fig. 6). HEV ORF3 antigen was detected from 7 dpi to 42 dpi. There was no significant change in HEV ORF3 protein expression from 7 dpi to 42 dpi. No positive HEV ORF3 antigen expression was found in any tissue from gerbils in control group. 4. Discussion Gerbils were successfully infected with HEV in this study. Our results showed that HEV RNA was detected in the livers of infected gerbils. The presence of HEV associated with hepatitis

was demonstrated using immunohistochemistry, and HEV ORF3 antigen was detected in infected livers. Although no clinical signs were observed, the liver-to-body weight ratios of inoculated gerbils were greater than those of control group. The liver enzyme levels in the experimental group were significantly higher than in the negative control group, which is consistent with previous reports (Kabrane-Lazizi et al., 1999; Huang et al., 2009; Li et al., 2009; Kasorndorkbua et al., 2003; Mao et al., 2014). Wild rats, which are trapped in many geographic areas, are HEV positive (Kabrane-Lazizi et al., 1999), as well as wild boar, deer, rabbits, and other animals (Worm et al., 2002). Because of its zoonotic characteristics, humans and many food animals, such as pigs and chickens are at risk of contracting HEV (Feagins et al., 2008; Huang et al., 2004). The Mongolian gerbil, which originates from the Mongolian desert and belongs to the subfamily Gerbillinae, is a common experimental gerbil species. Gerbils have also become popular pets in many countries (Kaplan and Hyland, 1972), increasing the risk of HEV transmission between humans and Mongolian gerbils. Therefore, HEV infection in Mongolian gerbil may be a potential public health risk. In our study, there was no evidence of clinical disease in the HEV experimental group. However, in a study by Yan Hong, Z:ZCLA Mongolian gerbils that were infected with human HEV exhibited fatigue

Fig. 3. Histopathological analysis of Mongolian gerbil livers. (A) No gross histopathology lesions were observed in liver sections from the control group. Lymphocytes infiltrations in the portal tracts of the experimental group were observed (). Varying levels of inflammatory cell infiltration were observed at different time points: (B) 7 dpi; (C) 14 dpi; (D) 21 dpi; (E) 28 dpi; (F) 42 dpi. H&E staining.


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Fig. 4. Ultrastructural pathological analysis of Mongolian gerbil livers. (A) There was no apparent liver damage. Electron density was high in the cell plasma (yellow arrow). (B) and (C) Spherical and elongated mitochondria with cristae were abundant and the cisternae of the endoplasmic reticulum were smooth (yellow arrow). (D) Electron density decreased in the cell plasma (blue arrow). (E) and (G) Mitochondria in the cytoplasm of the cell were swollen with thin cristae (blue arrow). (F) The cytoskeleton dissolved (blue arrow) and petals-like particles were observed in the cytoplasm (green arrow).

Fig. 5. Immunohistochemical analysis of Mongolian gerbils’ livers. (A) Histological liver sections from the control group at 28 dpi. (B) HEV ORF2 protein positive signal (→) expression in livers from the experimental group at 28 dpi. Immunohistochemical staining.


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0/6 N/A 0/6 N/A 0/6 N/A

6/6 5.15 ± 0.34b

0/6 N/A

6/6 6.17 ± 0.32

0/6 N/A

6/6 6.53 ± 0.18

0/6 N/A

6/6 7.73 ± 0.19

0/6 N/A

5/6 4.52 ± 0.37

0/6 N/A

Control Inoculated

a

b

Number of positive/number of total Data are means ± SD.

0/6 N/A

Number of HEV RNA positive liver Viral load in HEV RNA positive liver

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Fig. 6. HEV ORF3 antigen detection in the livers of Mongolian gerbils by Westernblotting. The expression of HEV ORF3 antigen was analyzed in livers from the experimental and control groups.

a

Inoculated Inoculated Inoculated Inoculated Inoculated Control

0 dpi

Inoculated Group

DPI

Table 3 HEV RNA detection in livers of experimentally infected gerbils.

7 dpi

Control

14 dpi

Control

21 dpi

Control

28 dpi

Control

42 dpi

Control

56 dpi


and loose hair (Hong et al., 2015). These findings may be the result of the differences in the two HEV strains used. The HEV strain we used to infected Mongolian gerbils was isolated from swine (genotype 4) and the HEV strain Yan Hong used to infect Z:ZCLA Mongolian gerbils was isolated from humans (genotype 1). The pathogenicity and the virulence of the two HEV strains may be different. The gerbils used were also not the same. The Mongolian gerbil and the Z:ZCLA Mongolian gerbil may exhibit difference responses to HEV infection. However, more research is necessary to confirm this. Liver weights in inoculated group increased significantly at 7 dpi, 14 dpi, 21 dpi, 28 dpi, and 42 dpi (p < 0.05). There was no obvious difference in body weight between the inoculated group and the control group. The liver-to-body weight ratios observed in the experimental group significantly increased at 7 dpi, 28 dpi, and 42 dpi (p < 0.05). This change was likely due to hepatic enlargement rather than the loss of body weight. In our study, we focused primarily on the liver, and conducted a further investigation resulting in pathological observations. In the Mongolian gerbil, viral hepatitis is characterized by lymphocyte infiltration in the portal tracts and hepatic lobules, which is consistent with Li’s results (Li et al., 2009), and may reflect the infectivity of HEV in Mongolian gerbils. Based on TEM observations of hepatocyte, the electron-density severely decreased, and the mitochondria were swollen in experimental animals, which may explain the vacuolized hepatocytes observed using light microscopy. These results are consistent with the increase in liver-to-body weight ratio observed during infection, and may be the cause of liver swelling. Swine HEV primarily damages the mitochondria, and further studies on the mechanism of HEV invasion of mitochondria are necessary. Many petal-like particles were also observed in the cytoplasm. The origin of these particles and the relationship between the particles and HEV replication in Mongolian gerbils are still unknown and require further investigation. According to a study by Yan Hong, histopathological changes can be observed in the liver, spleen and kidney of Z:ZCLA Mongolian gerbils infected with human HEV (Hong et al., 2015). This may indicate that the liver is not the only organ targeted by HEV infection. Moreover, further research on the effects of swine HEV infection on other organs, including the kidney, spleen, and brain are necessary. In our study, the virus was first detected in the liver at 7 dpi, which is not consistent with previous findings in pigs (3 days) (Williams et al., 2001) and is 7 days earlier than in nude mice (14 days) (Huang et al., 2009), but 3 days later than in laboratory rats infected with HEV (Maneerat et al., 1996). According to the ELISA results, the HEV antibody levels reached their highest level on day 42. However, the viral load of HEV RNA in the liver was lowest at 4.52 logs g−1 on 42 dpi and was highest at 7.73 logs g−1 at 28 dpi. This may be because HEV stimulates the immune system, decreasing antibody levels. The antibodies neutralize the antigen, such that the copy number of HEV RNA decreased at 42 dpi while, the HEV antibody was still at a high level. Levels of ALT, AST, and T-BIL increased by approximately twofold compared with baseline levels from 7 dpi to 56 dpi. These results are similar to results found in Balb/c nude mice (Huang et al., 2009). Animals were considered to be suffering from hepatitis


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when their serum ALT concentrations exceeded the pre-infection ALT levels by more than twofold (Zhang et al., 2003). Therefore, these results indicate that all of the gerbils from the experimental group were suffering from hepatitis. To the best of our knowledge, the current study is the first to report data on the viral load in the livers of Mongolian gerbils experimentally infected with swine HEV. Immunohistochemistry and Western-blotting results have shown that HEV antigen is expressed in the liver and demonstrated that one of the target tissues of HEV is the liver (Mao et al., 2014). The finding that HEV RNA replication in the liver is consistent with HEV ORF3 antigen expression by Western-blotting is evidence of HEV replication and HEV antigen expression in the livers of Mongolian gerbils (Williams et al., 2001). Combined with histopathological examination and ultrastructural analysis of the livers, these results demonstrate the presence of liver lesions in gerbils infected with swine HEV. In a previous study, using Balb/c nude mice, HEV RNA was detected in the liver from 4 to 14 dpi. In our study, HEV RNA was detected in the liver from 7 to 42 dpi (Huang et al., 2009). However, our results were more consistent with the last days of HEV RNA detection in a swine model (Bouwknegt et al., 2009). These results suggest that HEV RNA replication in the Mongolian gerbil model is similar to its replication in the swine model and that the Mongolian gerbil may be a suitable model for HEV research. The underlying mechanisms responsible for HEV replication at different time points and for the variable concentrations observed in the livers in this study require further investigation. Finally, this study is the first time systematically evaluates and reports the viral load in the liver of experimentally infected Mongolian gerbils. The underlying mechanisms for HEV replication at different time points and at different concentrations in the liver require further study. The combined results of histopathological examinations, ultrastructural analysis, liver enzyme concentrations, HEV ORF3 antigen expression in different tissues, and dynamic changes in HEV antibody levels in the serum support the finding of liver lesions following experimental HEV infection in Mongolian gerbils. These findings are important for future studies on the replication mechanism, transmission and pathogenesis of HEV. Competing interests The authors have declared no competing interests. Authors’ contributions YY and RShe were responsible for the study design. YY, RShi, MHS, JM, FD, and YZ did performed laboratory work. YY and CL analyzed the data. YY wrote the article. All authors read, commented on and approved the final article. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 31072110, 31272515). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. References Smith, D.B., Simmonds, P., Jameel, S., Emerson, S.U., Harrison, T.J., Meng, X.J., Okamoto, H., Van der Poel, W.H., Purdy, M.A., 2014. Consensus proposals for classification of the family Hepeviridae. J. Gen. Virol. 95 (Pt 10), 2223–2232. Emerson, S.U., Purcell, R.H., 2003. Hepatitis E virus. Rev. Med. Virol. 13 (3), 145–154.

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Swine is a possible source of hepatitis E virus infection by comparative study of hepatitis A and E seroprevalence in Thailand.

Induction of antibody and interferon-γ production in mice immunized with virus-like particles of swine hepatitis E virus.

One case of swine hepatitis E virus and porcine reproductive and respiratory syndrome virus co-infection in weaned pigs.

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Antiviral strategies for hepatitis E virus.