Hepatitis E Virus (HEV) strains in serum samples can replicate efficiently in cultured cells despite the coexistence of HEV antibodies: characterization of HEV virions in blood circulation

Abstract

We recently developed a cell culture system for hepatitis E virus (HEV) in PLC/PRF/5 and A549 cells, using fecal specimens from HEV-infected patients. Since transfusion-associated hepatitis E has been reported, we examined PLC/PRF/5 and A549 cells for the ability to support replication of HEV in various serum samples obtained from 23 patients with genotype 1, 3, or 4 HEV. HEV progenies emerged in culture media of PLC/PRF/5 cells, regardless of the coexistence of HEV antibodies in serum but dependent on the load of HEV inoculated (31% at 2.0 x 10(4) copies per well and 100% at >or=3.5 x 10(4) copies per well), and were successfully passaged in A549 cells. HEV particles in serum, with or without HEV antibodies, banded at a sucrose density of 1.15 to 1.16 g/ml, which was markedly lower than that for HEV particles in feces, at 1.27 to 1.28 g/ml, and were nonneutralizable by immune sera in this cell culture system. An immuno-capture PCR assay of HEV virions treated with or without detergent indicated that HEV particles in serum are associated with lipids and HEV ORF3 protein, similar to those in culture supernatant. By immunoprecipitation, it was found that >90% of HEV particles in the circulation exist as free virions not complexed with immunoglobulins, despite the coexistence of HEV antibodies. These results suggest that our in vitro cell culture system can be used for propagation of a wide variety of HEV strains in sera from various infected patients, allowing extended studies on viral replication specific to different HEV strains.

FIG. 1.

Quantitation of HEV RNA in culture supernatants of PLC/PRF/5 or A549 cells inoculated with serum samples, with or without HEV antibodies (passage 0) and at various HEV loads (10⁴ to 10⁶ copies per well) (a to e), or in culture medium of passage 0 (f). (f) The harvested culture supernatant of passage 0 was purified by passage through a microfilter with a pore size of 0.22 μm (see Materials and Methods) and then inoculated onto fresh A549 cells.

FIG. 2.

Phylogenetic tree constructed by the neighbor-joining method, based on the partial nucleotide sequence of the ORF2 region (412 nt) of 64 HEV isolates, using an avian HEV (GenBank accession no. AY535004) as an outgroup. In addition to 39 HEV isolates obtained in the present study, which are indicated in bold, 24 representative HEV isolates of genotypes 1 to 4 whose common 412-nt sequence is known were included for comparison, and their accession numbers, followed by the names of the countries where they were isolated, are indicated. “wt” denotes an HEV isolate recovered from a serum sample that was used to inoculate cells, and “p0” represents an HEV isolate obtained from culture supernatant harvested 30 days after inoculation of the corresponding serum sample. Bootstrap values are indicated for the major nodes as percentages of the data obtained from 1,000 resamplings. Bar, 0.1 nucleotide substitution per site.

FIG. 3.

(a) Quantitation of HEV RNA in culture supernatants of A549 cells inoculated with a serum sample (S1) with a viral load of 5.0 × 10⁵ copies/ml that had been mixed with a pooled serum sample obtained during the early convalescent phase (pool 1, 2, or 3) and positive for IgM-, IgA-, and IgG-class anti-HEV antibodies or with an antibody-negative serum and cultured for 50 days. (b) Quantitation of HEV RNA in culture supernatants of A549 cells inoculated with a serum sample (S1) with a viral load of 5.0 × 10⁵ copies/ml that had been mixed with an anti-ORF2 MAb (H6225) (51), anti-ORF3 MAb (TA0536) (52), or negative-control MAb (905) (46) at a final concentration of 1 mg/ml and cultured for 50 days. (c) Quantitation of HEV RNA in culture supernatants of A549 cells inoculated with a fecal suspension (JE03-1760F), a serum sample (S4a), or a culture supernatant of JE03-1760F (patient 4) origin with an HEV load of 2.0 × 10⁵ copies/ml that had been mixed with a serum sample (03-2150) positive for IgM-, IgA-, and IgG-class anti-HEV antibodies but negative for HEV RNA, another serum sample (08-1340) positive only for IgG anti-HEV, or an antibody-negative serum and cultured for the indicated number of days. The fecal suspension, serum sample (S4a), and culture supernatant contained HEV of the same strain (JE03-1760F), and serum samples (S4a, 03-2150, and 08-1340) were obtained from the JE03-1760F patient (patient 4 in this study) 15, 40, and 2,088 days, respectively, after disease onset.

FIG. 4.

Sucrose density gradient fractionation of HEV in a fecal specimen (JE03-1760F) and in a culture supernatant containing cell culture-produced HEV, as controls, and in serum samples with (S6 and S7) and without (S1 and S2) HEV antibodies.

FIG. 5.

Comparison of HEV RNA titers in culture supernatants of cultured cells obtained during the observation period of 60 days after inoculation between an original serum sample (S7) and its peak fraction in a sucrose density gradient, between an original culture supernatant and its peak fraction in a density gradient, and between an original fecal suspension (JE03-1760F) and its peak fraction in a density gradient (Fig. 4). The loads of HEV inoculated were 1.0 × 10⁴ copies per well for the serum sample and its peak fraction, 1.0 × 10⁵ copies per well for the culture supernatant and its peak fraction, and 1.2 × 10⁴ copies per well for the fecal specimen and its peak fraction.

FIG. 6.

Sucrose density gradient fractionation of HEV in culture supernatants treated with 5% Tween 20, 10% chloroform, or a solution containing 0.1% NP-40 and 0.1% pronase E.

FIG. 7.

Quantitation of HEV RNA in culture supernatants of A549 cells inoculated with a culture supernatant with a viral load of 1.0 × 10⁵ copies/ml that had been treated with or without 0.1% NP-40 and 0.1% pronase E, mixed with a normal serum or pooled serum sample obtained at the early convalescent phase (pool 1) and positive for IgM-, IgA-, and IgG-class anti-HEV ORF2 and ORF3 antibodies, and cultured for 30 days.