Cell Culture of Sporadic Hepatitis E Virus in China

Virus strains.Four virus strains, designated G93-1, G93-2, G93-3, and G93-4, were originally isolated in the A549 cell line from the feces of four patients with acute HE at Guangzhou Municipal Infectious Diseases Hospital in 1993. Detailed information has been reported elsewhere (17).

Cells.The A549 cell line was prepared in aseptic bottles. The cells were grown in mixed medium (50% medium 199 and 50% Dulbecco modified Eagle medium) supplemented with 10% heat-inactivated calf serum, 100 U of penicillin per ml, 100 μg of streptomycin per ml, and 50 U of kanamycin per ml at 37°C. The medium used for virus culturing was 5 ml of maintenance medium containing 2% inactivated calf serum and 30 mM MgCl₂ (final concentration); other components were the same as in growth medium (9). In addition, strain 2BS, a diploid strain of human fetal lung fibroblasts, was obtained from the Beijing Institute of Biological Products and used for passages 26 to 33 in this study. The LLC-MK₂ continuous cell line was also used. Culture media and methods used for these cells were the same as those described elsewhere (9).

Antibodies.Anti-HEV rabbit serum and mouse ascitic fluid were prepared in our laboratory. Titers of polyclonal antibodies obtained by 4 weeks postimmunization were 1:640 and 1:2,560, and those of monoclonal antibodies (e.g., B₄C) were 1:1,600 and 1:12,800, as determined by an indirect immunofluorescence assay and an enzyme-linked immunosorbent assay, respectively.

Virus passage.Briefly, viruses were inoculated onto a monolayer of A549 cells at a multiplicity of infection (MOI) of 0.025, and then maintenance medium was added. The inoculated cell monolayer was incubated at 37°C and observed for cytopathic effect (CPE) daily. When the CPE reached +++, both cells and supernatants were harvested and stored.

Virus titration.After serial 10-fold dilutions were made in growth medium, 0.025 ml of each different dilution was inoculated into four wells each containing 0.025 ml of growth medium, and then 0.5 ml of a cell suspension was added. The cultures were incubated at 37°C in a humidified 5% CO₂ atmosphere and observed for CPE daily for 7 days. Viral titers were calculated by the method of Reed and Muench (13a) and expressed as 50% tissue culture infective doses (TCID₅₀)/0.025 ml.

Conditions for virus culturing and the cell sensitivity test.To select the best propagation conditions, we used an L9(3⁴) positive-cross design. Three factors and three levels were selected to conduct the test: the final concentration of Mg²⁺ was 0, 30, or 60 mM; the pH value was 6.8, 7.2, or 7.6; and the inoculum dose had an MOI of 0.25, 0.025, or 0.0025. The cultures were incubated at 37°C for 72 h and observed for CPE, and viral titers were determined by microculturing after the cell suspension had been frozen and thawed. The results were analysed with SAS software.

For a comparison of sensitivity in different cells, viruses were inoculated into 2BS, A549, and LLC-MK₂ cells at an MOI of 0.025. The cells were cultivated in series for three generations and harvested when the CPE reached +++ or on day 7 if the cells were negative for CPE. Viral titers prepassage and postpassage were determined with A549 cells.

Physical and chemical tests.Passage 8 suspensions of strains G93-1, G93-2, G93-3, and G93-4 were tested for resistance to acid (pH 3.0), ether, and heat (56°C) and for nucleic acid type. The methods used were the same as those reported previously (19).

Electron microscopy.Passage 4 of HEV strain G93-2 was inoculated into A549 cell monolayers. Thin sections were prepared as previously described (7). Briefly, HEV strain G93-2 was inoculated into A549 cells at 0.1 ml of virus stock per flask. After the appearance of CPE in infected cell cultures, the medium was harvested, frozen-thawed five times, and centrifuged at 2,600 ×g for 40 min to remove debris. After centrifugation (6,000 × g, 30 min), the suspension was precipitated with 8% polyethylene glycol (PEG) at 4°C overnight. The bottom sediment was suspended in phosphate-buffered saline. The viral suspension was incubated at 37°C for 1 h with an equal volume of ascitic fluid (1:8 or 1:16) and then at 4°C overnight, with centrifugation (12,500 × g, 30 min) pre- and postincubation. The virus-antibody complex was suspended once more in pure water, applied to carbon-coated grids, negatively stained with 2% phosphotungstate (pH 7.2), and observed and photographed with a Philips CM 120 electron microscope (12).

Identification of the partial genome.Cell cultures (10 ml) of strains G93-1, G93-2, G93-3, and G93-4 (passage 5) were concentrated to 0.1 ml with PEG. RNA was extracted with TRIzol reagent (Gibco BRL) according to the manufacturer’s instructions as previously described (13). After denaturation for 10 min at 70°C, reverse transcription and PCR were performed. Two sets of sense and antisense synthetic oligonucleotide primers for the HEV ET1.1 region were used for detection of the HEV genome. The sequences of the primers used in this study were as follows: F₁, 5′-GCT ATT AGT GAG GAG TGT GG-3′ (positions 4459 to 4478); R₁, 5′-CAG GGC CCC AAT TCT TCT-3′ (positions 4876 to 4859); F₂, 5′-GCG TGG ATC TTG CAG GCC-3′ (positions 4522 to 4539); and R₂, 5′-TTC AAC TTC AAG CCA CAG CC-3′ (positions 4760 to 4741). The other reactions were the same as those previously described (10). The PCR products were analyzed by 1.5% agarose gel electrophoresis. In order to confirm the specificity of the segments amplified by PCR, Southern blotting was carried out with a 239-bp DNA fragment obtained from HEV strain 87A as a probe. The recovered DNA was ligated with the pGEM-T Easy Vector (Promega) and transformed in JM109. Positive clones were screened by PCR and identified with EcoRI and HindIII. Positive recombinants were analyzed with an ABI model 373A DNA sequencer. The homology of this part of the nucleotide sequence was compared among strains G93-1, G93-2, G93-3, and G93-4 and Xinjiang strain 87A (7).