Hepatitis E virus in Norway rats (Rattus norvegicus) captured around a pig farm

Animals

Norway rats (Rattus norvegicus) were caught, using commercial snap traps at 6 different locations, around a pig farm where HEV genotype 3 was prevalent among pigs (Hokkaido, Japan) [36]. The farm consists of three buildings. The capture locations surrounded 3 different buildings where the grow-finishing pigs (about 120 total head counts) were reared. Our previous study revealed that all the pigs were infected with HEV genotype 3. These studies were performed in accordance with the guidelines for the capture, handling, and care of mammals of the Mammalogical Society of Japan. All animal experiments were approved by the Rakuno Gakuen University Ethical Committee for Animal Experiment Regulation, Hokkaido, Japan (approved #VH21C10).

The power analysis used to calculate the number of animals required for the study was performed on the basis of the 95% confidence interval shown on the Raosoft^® website (http://www.raosoft.com/samplesize.html). Because the prevalence rate of HEV infection in wild rodents was estimated to be approximately 10% [28–31], the values required for calculating the sample size were set as follows: infection rate, 10%; margin of error that determines the range of the 95% confidence interval, 5%; and confidence level that refers to the likelihood of 95% confident interval, 90%. The power analysis indicated the appropriate sample size to be 97 to obtain 10% HEV infection in rats with a 5% margin of error. Since we found a sufficient number of HEV infections in rats when 56 animals were examined, no further capturing was performed.

Sampling and RNA extraction

The liver, spleen, intestines, and blood were collected from 56 wild rats to determine the presence of HEV RNA. Tissue and serum samples were collected from each rat, and the samples were stored at -80°C until analysis. Blood was collected from the hearts of dead wild rats by using filter paper (Toyo Roshi; Advantec, Tokyo, Japan) according to the manufacturer's instructions. The filter paper was dissolved in 1 mL of phosphate-buffered saline (PBS) and subsequently diluted (1:25) to make serum samples. During tissue collection from wild rats, each dissection instrument was sterilized to avoid contaminating tissues with HEV RNA. Tissue samples (100 mg) were homogenized using zirconia beads with a TissueLyser (Qiagen GmbH, Hilden, Germany). Viral RNA was extracted from 100 mg of the tissue sample by using 1 mL of TRIzol reagent (Life Technologies Corp., Carlsbad, CA) according to the manufacturer's instructions. The final elution was carried out using 50 μL of RNase-free H₂O. The RNA extracted from HEV-infected (genotype 3) swine livers was used as a positive control.

ELISA

Anti-HEV antibodies were detected by ELISA with a commercial kit (Viragent HEV-Ab kit; Cosmic Corporation, Tokyo, Japan) that used a truncated recombinant HEV ORF2 protein expressed in silkworm pupae [37] according to the manufacturer's instructions. For the positive control serum, 3 Wistar rats were subcutaneously immunized with ORF2 antigen (100 μg/rat) 3 times every 2 weeks. The recombinant ORF2 protein was produced as a fusion protein with glutathione-S- transferase (GST) from the plasmid, pGEX-AC2.1, which encodes the ORF2 antigen (genotype 3). TALON Metal Affinity Resin (Clontech Inc., Palo Alto, CA) was used to purify the recombinant ORF2 protein. The sera from 5 intact Wistar rats were used as negative controls. The serum samples were diluted in buffer (1:100) and were incubated for 1 h at room temperature. Because the ELISA kit was developed for detecting human antibodies, an HRP-conjugated anti-rat IgG antibody (Zymed Inc., South San Francisco, CA) was used as the secondary antibody. After the secondary antibody reactions, 50 μL of TMB (3,3',5,5'-tetramethylbenzidine) (Kirkegaard & Perry Laboratories Inc., Baltimore, MD) was added; after 30 min incubation at room temperature, 50 μL of 2 M sulfuric acid was added to stop the reaction. The optical density at 450 nm (OD₄₅₀) was measured. The cutoff value for IgG ELISA was calculated as the mean OD + 3 SDs of 5 uninfected Wistar rats (cutoff: OD 0.3).

RT-PCR and sequence analysis

Initially, the HEV RNA of the 5' terminal region of ORF1 was detected by semi-nested RT-PCR [36] with the sense primer HE61 (5'-CACRTATGTGGTCGAYGCCATGGAG-3'; R = A or G, Y = C or T) and the antisense primer HE51 (5'-GCCKRACYACCACAGCATTCG-3'; K = G or T) for reverse transcription (RT) and first round of PCR and the internal sense primer HE50 (5'-AAGGCTCCTGGCRTYACWAC-3'; W = A or T) for the second round of PCR to confirm the first round of PCR products. Reverse transcription and first-round amplification were carried out using the OneStep RT-PCR Kit (Qiagen). In each reaction, 5-μL aliquots of viral RNA solution were used. The reactions were performed in an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany) under the following conditions: RT at 50°C for 30 min, denaturation at 95°C for 15 min; 45 cycles of denaturation at 95°C for 15 s each, annealing at 55°C for 30 s, and elongation at 72°C for 30 s; and final extension at 72°C for 7 min. After the first round of PCR, 1-μL PCR product was amplified under the following conditions: 20 cycles of denaturation at 95°C for 15 s each, annealing at 60°C for 30 s, and elongation at 72°C for 15 s, followed by a final incubation at 72°C for 7 min. The amplified second-round PCR products were confirmed by 2% gel electrophoresis. The expected amplicon sizes of ORF1 were 125 bp and 85 bp in the first- and second-round PCRs, respectively. The RT-PCR-positive samples were also confirmed by semi-nested RT-PCR for the ORF 2 region by using the sense primer HE169 (5'-GAGGAGGAGGCTACTTCCG-3') and the antisense primer HE 171 (5'-CAGCCGACGAAATCAATTCTGTCG-3') for the RT and first-round PCR, and for the semi-nested PCR primer HE170 (5'-GTAATGCTTTGCATTCACGGCTCC-3'). The expected amplicon sizes of ORF2 for the first- and second-round PCRs were 373 bp and 349 bp, respectively. A 349-bp PCR amplicon was determined to be specific to HEV.

The PCR products from 7 positive samples (3 spleens and 4 intestines from 6 rats captured at 6 different locations) were excised from the gel, purified using QIAquick Gel Extraction Kit (Qiagen) and sequenced to confirm the specificity of the RT-PCR reaction. The purified products were cloned into a plasmid (pTA2, Cat: TAK-101; Toyobo Co. Ltd., Osaka, Japan) and sequenced using the M13 forward and reverse primers. The genomic sequences of the 5' terminal region of HEV ORF1 were compared using Bio Edit (version 7.0.9.0) (http://www.mbio.ncsu.edu/BioEdit/bioedit.html).

Virus isolation from wild Norway rats

To isolate infectious rat HEVs, both the spleens and intestines of 3 of the HEV-positive wild rats (#38, #49, #50) were homogenized, filtered through a 0.22-μm Millipore filter (Millipore, MA), and inoculated into A549 cells (human alveolar basal epithelial cells), which are sensitive to HEV infection [38–40]. After adsorption for 60 min, the cells were washed with PBS and cultured in DMEM (Sigma-Aldrich Corp., St. Louis, MO) containing 2% fetal bovine serum and insulin-transferrin-selenium-X supplement (Cat. 51500-056; Life Technologies Corp., Carlsbad, CA) at 37°C in a 5% CO₂ incubator [40]. The cells were passaged 3 days post-infection (dpi) and cultured in the same medium conditions. At 7 dpi, viral RNA was extracted from culture supernatants and cells by using a QIAamp Viral RNA Mini Kit (Qiagen). HEV RNA was examined by semi-nested RT-PCR for the ORF1 region as described above.

Immunofluorescence assay

HEV antigen was detected by immunofluorescence assay to confirm the infectivity of rat HEV. Virus-infected cells (7 dpi) fixed with acetone-methanol for 5 min were washed with PBS and reacted with pig anti-HEV polyclonal antibody purified from pigs infected with HEV genotype 3 [41]. After incubation for 60 min at room temperature, the cells were washed with PBS containing 0.1% Tween 20 and reacted with FITC-rabbit anti-porcine IgG (Zymed) for 60 min at room temperature. After washing with the buffer, stained cells were examined under a Zeiss Pascal confocal microscope (LSM 5; Carl Zeiss AG, Oberkochen, Germany).