Clinical sample collection and processing
In December 2020, one hundred rectal swabs were collected from Bama miniature pigs at a laboratory animal center in Sichuan Province, southwest China. Each sample was diluted in phosphate-buffered saline to generate a 10% (w/v) fecal homogenate suspension, followed by clarification of suspensions via centrifugation at 4500 × g for 10 min at 4 °C.
Amplification of the partial ORF1 and ORF2 genes of HEV
200 μL of supernatant was used for total RNA extraction by TRIzol Reagent (TaKaRa, China). All samples were analyzed using a broad-spectrum nested reverse transcription polymerase chain reaction (RT-nPCR) with specific primers designed to amplify the partial HEV ORF1 gene, which were described previously by Reimar Johne . In addition, to confirm detection of HEV, the partial ORF2 gene of the HEV genome was also amplified using RT-nPCR as described previously . All purified positive PCR products were sequenced by Genetic Analyzer（ABI 3130, Applied Biosystems, USA). Two sequences were submitted to GenBank (Accession numbers MW498242 and MW498243).
Based on sequences that were obtained, multiple alignments were performed using the MegAlign program (DNASTAR Inc., Madison, WI). Next, phylogenetic trees were constructed that also incorporated other known GenBank HEV strain sequences using the MEGA7 software. GenBank numbers included D10330, D11092, D11093, KX578717, M74506, FJ527832, AY115488, AP003430, AB197673, EF077630, EU366959, KC492825, MK410045, DQ279091, EU676172, AB074915, AB200239, AY594199, FJ610232, GU206559, GU361892, KF176351, AY723745, AB220974, AB108537, GU119961, GU188851, AB369690, DQ450072 and HM439284.
Animal experiment design and samples collection
Six healthy Bama miniature pigs (body weight, 5 kg) negative for HEV RNA and anti-HEV antibodies were randomly divided into two groups of 3 pigs each. The virus was quantitatively analyzed by RT-nPCR as previously described  and the titer of this infectious stock was 106 genome equivalents per ml (106 GE/ml). A total of 500 μL of this stock was inoculated intravenously into each pig in the experimental group, and the three pigs in the other group served as negative controls. Fecal and serum samples were collected from each pig before inoculation and weekly. Serum samples were detected for alanine aminotransferase (ALT) levels and anti-HEV antibodies. Fecal and serum samples were also tested for HEVs RNA by RT-nPCR. After pigs were necropsied at 10 week post inoculation (wpi), liver samples were collected and fixed in 10% neutral buffered formalin for histological examination and immunohistochemistry (IHC).
Detection of anti-HEV antibodies and ALT concentrations
Anti-swine HEV IgG antibodies were tested in serum samples by indirect ELISA as previously described . Briefly, purified CHN-SD-sHEV truncated capsid protein (200 ng/well) was coated on the plates overnight at 4°C. After blocked and washed, serum samples (1:100, 100 μL/well) were added into each well and incubated for 1 hour at room temperature (RT). After washed, horseradish peroxidase (HRP)-conjugated goat anti-swine IgG (Jackson ImmunoResearch, West Grove, PA, USA) (1:5000, 100 μL/well) was added and also incubated for 1 hour. After washed again, 3,3´,5,5´-tetramethylbenzidine (TMB) was added and the plates were incubated in the dark for 15 min at RT. The colorimetric reaction was stopped (3 M H2SO4, 50 μL/well) and optical density (OD) values were read at 450 nm by an automated microplate reader (Bio-Rad, USA). Each sample was detected in duplicate wells.
ALT concentrations in plasma samples from pigs were measured using standard methods. Pigs were considered positive for hepatitis, when their ALT levels exceeded pre-challenge ALT levels more than two-fold .
Detection of swine HEV RNA using RT-nPCR
Swine HEV RNA isolated from fecal and serum samples from inoculated pigs were tested according to the method described previously . Briefly, total RNA was extracted from 10 % fecal suspension or 200 μL sera by TRIzol Reagent according to the manufacturer’s recommendations. For RT-nPCR, reverse transcription and first PCR were performed using PrimeScript™ One Step RT-PCR Kit (TaKaRa, China). Next, the second PCR was conducted using TransTaq High Fidelity DNA polymerase (TransGen Biotech, China) based on the manufacturer’s instructions. Finally, PCR products were identified by electrophoresis on 1% agarose gel.
Evaluation of histopathological and immunohistochemical changes in liver tissues
During necropsies, the liver tissues were harvested separately and fixed for routine histological examination. IHC analyses were conducted using an UltrasensitiveTM SP kit and a DAB Detection Kit (Fuzhou Maixin Biotechnology Development Co., China) based on the manufacturer’s instructions. The monoclonal antibody 3E8 (mouse anti-HEV capsid protein, 1mg/ml, 1:1,000 dilution) was used.
Data collection and analyses were conducted by IBM SPSS Statistics 20 (IBM, USA). Student’s t-test was used to analyze the ALT values between different groups (P ≤ 0.05 was considered significant).