Human lung carcinoma A549 cells were obtained from the American Type Culture Collection (ATCC^® CCL-185^TM) and were grown at 37 °C with 5 % CO₂ atmosphere in Dulbecco's modified Eagle's maintenance medium (DMEM, 100 U/mL penicillin, 100 mg/mL streptomycin and 5 % fetal bovine serum). The fecal sample was kindly donated by Servicio de Trasplante Hepático del Hospital Central de las Fuerzas Armadas, Montevideo, Uruguay, and it was obtained from a human feces sample of a South American patient in the acute phase. The sample was previously characterized extracting RNA (TRIzol, LifeTechnologies) from 1 mL of a 10 % suspension of fecal material in phosphate-buffered saline (PBS, Gibco^® No Calcium, No Magnesium). To detect the viral genome, an RT-PCR was performed with the 5' end of ORF1 as the target, according to Mirazo et al., (2013). For genotyping, a phylogenetic analysis was implemented using the Maximum-Likelihood method and the Tamura-Nei evolutionary model, using MEGA v6 (Mainardi et al., 2018, submitted).

For virus isolation, 50 - 70 % -confluent A549 cell monolayers grown in six-well plates (Falcon^®) were washed three times with 1 mL PBS. The fecal sample was diluted 2-fold in PBS and filtered through acrodisc syringe filters with a pore size of 0.22 μm (Millex-GV; Millipore Corp., Bedford, MA). A549 monolayers were infected with 0.5 mL of the filtrated sample containing 5.85 x 10⁵ RNA copies/µL per well at 37 °C and 5 % CO₂ atmosphere. After two hours the inoculum was removed, cells were washed three times with PBS, and then 2.5 mL of cell maintenance medium was added to each well and incubated at 37 °C in a humidified 5 % CO₂ atmosphere. Four days post-infection (dpi) cells were passaged for the first time. Cells were washed, dispersed with 0.25 % trypsin (500 μL), diluted 4-fold in fresh maintenance medium, and 1 mL was added to a new cell well. Infected cells were thus passaged serially and tested by RT-PCR every 3 - 5 days for 41 days. The infected cell cultures were examined daily under an inverted microscope (Nikon TS 100 F), but no specific cytopathic effect was observed. Viral stocks were obtained from infected monolayers by two freeze-thaw cycles in liquid nitrogen, and viruses were quantified by determining the viral genome copy number by RT-qPCR and stored at -80 °C until use.

For total RNA isolation, TRIzol (Invitrogen^®) was used according to the manufacturer's directions. Then, RT-PCR was performed targeting a region within the ORF1 (partial methyltransferase gene and hypervariable region) as previously described (Mirazo et al., 2013). For RT-qPCR we used the forward (JVHEVF; 5'-GGTGGTTTCTGGGGTGAC-3') and reverse primers (JVHEVR; 5'-AGGGGTTGGTTGGATGAA-3') targeting a region within the ORF3 previously reported (Jothikumar et al., 2006), and the 25 μL mixtures were incubated at 42 °C for 30 minutes, followed by 95 °C for 10 minutes, and 39 cycles at 95 °C for 20 s, 60 °C for 20 s, and 72 °C for 45 s. A melting curve was run to verify the specificity of the products and was performed at 95 °C for 15 s, 60 °C for 15 s and 95 °C for 15 s, collecting data every 0.2 s. A 10-fold serial dilution of the RNA standards (10²10⁸ copies) was used for the quantitation of viral genome copies numbers (Mirazo et al., 2018). The RT-qPCR analysis was performed in the ABI 7500 Real-Time PCR System. All procedures were performed by triplicate and data are expressed as means (±S.D.).

To test if viral stock obtained from infected monolayers by two freeze-thaw cycles in liquid nitrogen could be able to infect fresh monolayers, 1 x 10⁵ A549 cells were seeded in 24 well plates (Falcon^®), 24 hours before viral infection. Then, cells were treated during two hours at 37 °C with two different inoculum concentrations obtained from cell lysates prepared in serum-free media (1 x 10⁴ or 1 x 10⁵ RNA copies/µL per well, by duplicate in two independent experiments). After that, the viral inoculum was removed and 500 μL of maintenance medium was added to each well. 96 hours post-infection (hpi), viral RNA and antigen detection were done by RT-qPCR and immunofluorescence assays (IFA), respectively. For IFA, cells were fixed with 200 μL per well of 75 % ethanol for 15 minutes; then cells were washed twice with PBS and treated with 200 μL of 0.1 % triton X-100 prepared in PBS. After blocking non-specific binding sites with 1 % bovine serum albumin (BSA) diluted in PBS, a 1:200 dilution of commercial monoclonal antibody against capsid antigen was added (ABCam, UK). Antibody was incubated for one hour at 37 °C and then washed twice in BSA-PBS prior incubation with FITC-labelled secondary antibody (goat anti-mouse IgG ABCam) for one hour at 37 °C. After incubation with the secondary antibody, cells were washed and DAPI diluted 1:100 in PBS was added for five minutes. Cells were rewashed and examined for the presence of HEV antigen by using an inverted fluorescence microscope (Olympus IX81, filters U-MNUA2, 360 - 370 nm, and U-MNIBA3 470 - 495 nm).

HEV positive A549 cell cultures were found by RT-PCR after 26 dpi with serial passages every three to four days (Figure 1a). The viral concentration of each day of sampling was quantified. The highest RNA copies number was found after 26 dpi (2 x 10⁶ RNA copies/µL) and viral RNA concentration decrease after day 35. No specific cytopathic effect was observed, and the evaluation finished on day 41 (Figure 1b). Capsid antigen and HEV RNA was detected in A549 cells infected with the isolated virus HEV is a highly tricky virus to isolate and for this reason, we tested if the viral stock isolated after 26 dpi were infectious to new A549 cells. Cells were treated with two viral concentrations prepared in serum-free media (1 x 10⁴ and 1 x 10⁵ copies/µL), and after 96 hpi, capsid antigen was detected perinuclearly and forming foci on A549 cells infected with 1 x 10⁴ copies/µL (Figure 2a) and viral RNA was detected in cell monolayers but no in supernatants. Of note, no statistically significative difference in viral genome copy number between the two viral concentrations used was found (p = 0.68 Mann-Whitney. U-test) (Figure 2b), indicating that this dilution was infectious.

Figure 1

Figure 2