PMC

Schematic representation of HAV vector constructs. A polylinker coding for SalI, SnaBI, and KpnI restriction sites flanked by HAV protease 3C^pro cleavage sites and Gly hinges was cloned at the 2A-2B junction of the HAV cDNA in pHAV8Y and termed pHAV8Y-MCS. The blasticidin resistance bsd gene without translation and initiation codons was cloned into the SalI and KpnI sites of pHAV8Y-MCS. The resultant construct, termed pHAV8Y-Bsd, carried the bsd gene in frame with the polyprotein, and its gene product Bsd should be released from the polyprotein by 3C^pro cleavage.

IF analysis of wt HAV rescued from transfected Huh7 cells. Huh7 cells were mock transfected (A) or transfected with HAV8Y-Bsd (B) or HAV.WT-Bsd (C) in vitro-synthesized RNA, selected with 1 μg/ml blasticidin for 2 weeks, and grown in eight-well slides. FRhK-4 cells were mock infected (D) or infected with HAV8Y-Bsd (E) or HAV.WT-Bsd (F). Huh7 cells were mock infected (G) or infected with HAV8Y-Bsd (H) or HAV.WT-Bsd (I). Infected cells were incubated for 2 weeks without selection and then grown in eight-well slides in the absence of blasticidin. Cells were fixed with cold acetone and stained with anti-HAV MAbs K2-4F2 and K3-4C8 and fluorescein isothiocyanate-conjugated goat anti-mouse antibodies. Immunofluorescent micrographs were taken with a Zeiss Axioscope microscope at 400× magnification using an oil-immersion objective.

Huh7 cells cured of HAV8Y-Bsd infection. (A) IF analysis. Huh7 cells mock infected (Mock), HAV8Y-Bsd infected (HAV8Y-Bsd), or HAV8Y-Bsd infected and cured by treatment with 250 U/ml IFN-αA/D (Cured) were grown in eight-well slides and stained with anti-HAV antibodies for IF analysis as described for Fig. . (B) Susceptibility of Huh7 and interferon-cured Huh7-A-I cells to infection with HAV8Y-Bsd and HAV.WT-Bsd. Titers were assessed in both cell lines using the blasticidin resistance endpoint titration assay as described for Fig. . (C) Susceptibility of Huh7 and interferon-cured Huh7-A-I cells to infection with wt HM-175 HAV derived from human stools. Cell lines were infected with HAV8Y-Bsd as a positive control. Titers were assessed by the ELISA endpoint titration assay. The titration was repeated at least three times with similar results, and the values represent one titration. Values are log₁₀ of the HAV titers determined by the Reed and Muench method (), and the standard deviations are shown as lines.

Growth of wt HAV in different cell lines. (A) One-step growth curve analysis of wt HAV recombinant viruses containing the Bsd selectable marker (HAV8Y-Bsd and HAV.WT-Bsd), wt HAV isolated from human stools (wt HM-175 HAV), and cell culture-adapted HAV (HAV/7) was done in monkey kidney (FRhK-4) cells, human hepatoma (Huh7) cells, and interferon-cured (Huh7-A-I) cells. Viral titers were assessed in Huh7-A-I cells using the ELISA endpoint titration assay as indicated in Fig. . (B) Schematic representation of nucleotide sequences. Full-length nucleotide sequences of HAV8Y-Bsd, HAV.WT-Bsd, wt HM-175 HAV, and HAV/7 RNA extracted from the last time point sample of the growth curve in Huh7-A-I cells were obtained by RT-PCR. Sequences that differ from the wt HM-175 HAV are indicated with the nucleotide position under the viral genomes. The main cell culture-adapting mutation at nt 3889 is indicated in boldface. Viral genes, the IRES, and the 3′-end poly(A) tail are indicated. The bsd gene clone in HAV8Y-Bsd and HAV.WT-Bsd is shown as a gray box.

Stability of wt HAV in Huh7 cells. (A) IF analysis of Huh7 cells infected with HAV8Y-Bsd or HAV.WT-Bsd and selected with 2 μg/ml blasticidin for 2 weeks. Mock-infected Huh7 cells grown in the absence of blasticidin were used as a negative control (Control). Cells were grown in eight-well slides and stained with anti-HAV antibodies as indicated in Fig. . (B) Titers of HAV8Y-Bsd and HAV.WT-Bsd grown in Huh7 cells were assessed in Huh7 and FRhK-4 cells using the blasticidin resistance endpoint titration assay in the presence of 2 μg/ml blasticidin. Plates were observed under the microscope 7 days after infection, and wells containing surviving cells were counted as positive for the titer determination. The titration was repeated at least three times with similar results, and the values represent one titration. Values are log₁₀ of the HAV titers determined by the Reed and Muench method (), and the standard deviations are shown as lines.