Fetal Calf Serum Exerts an Inhibitory Effect on Replication of Duck Hepatitis A Virus Genotype 1 in Duck Embryo Fibroblast Cells

Figures

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  Figure 1. Growth kinetics of DHAV-1 in DEF cells. The fifth cell passage viruses of C-QYD and C80 were used to inoculate DEF cells at a MOI corresponding to one copy/cell. Viral yields in DEF cells were reflected by RNA copies detected with RT-qPCR. At each time point, cells + supernatant (A) and supernatant only (B) were respectively collected from three wells of 24-well plates. Error bars represent the SD (n = 3); * p < 0.05.

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  Figure 2. Characterization of DHAV-1 growth in DEF cells. (A) DEF cells after inoculation with DHAV-1. Upper panel, the third cell passage of C-QYD and the second cell passage of C80 were used as inocula. Two percent CS DMEM served as maintenance medium. The cells were checked at 24 hpi. Lower panel, the fifth passages of C-QYD and C80 were used as inocula. Two percent FCS DMEM served as maintenance medium. The cells were checked at 72 hpi. (B) Immunofluorescence showing in DEF cells 24 h after inoculation with the fifth cell passage viruses of C-QYD and C80. Upper panel, 2% CS DMEM served as maintenance medium; lower panel, 2% FCS DMEM served as maintenance medium. The analysis was conducted by using specific chicken antisera against C-QYD and a FITC-conjugated goat anti-chicken IgY, followed by nuclear staining with DAPI. Bar = 50 μm. (C) Titers of the first to fifth cell passage viruses of C-QYD and C80. DEF cell cultures 24–72 h after inoculation with virus were sampled and tested by using RT-qPCR assay. Error bars represent the SD (n = 3); ** p < 0.01.

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  Figure 3. Effect of serum type on formation of plaques in DHAV-1-infected DEF cells. (A) DHAV-1 plaques in DEF cells overlaid with agarose overlays containing CS and FCS, respectively. Plaques were visualized by crystal violet staining at 60 hpi. (B) Comparison of the effect of CS and FCS on diameters of DHAV-1 plaques in DEF cells. ND: Not detectable.

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  Figure 4. Quantitation of DHAV-1 genome replication in DEF cells inoculated with serum–virus mixture. The DHAV-1 C-QYD and C80 strains were individually preincubated with three concentrations (0.5%, 1%, and 2%) of FCS (A,B) and CS (C,D) at 37 °C for 1 h. DMEM–virus mixture was included as control. For each serum concentration, samples (cells + supernatant) were collected from three wells of 24-well plates. Viral load in each sample was quantified by RT-qPCR. Error bars represent the SD (n = 3); * p < 0.05; ** p < 0.01.

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  Figure 5. Investigation into the effect of FCS on virus adsorption and penetration. (A) Virus attachment inhibition assay. The FCS–virus mixture was used as inoculum. After adsorption at 4 °C for 1 h, the cells were washed to remove unbound virus, and the amount of bound virus was quantified. (B) DEF cells were pretreated with 2% FCS DMEM at 37 °C for 1 h, followed by inoculation with virus. Viral loads in cells + supernatant were detected at 18 hpi. (C) Analysis of effect of FCS on virus entry. Virus adsorption was carried out at 4 °C, followed by incubation of the cells with FCS at 37 °C for 1 h. Proteinase K was employed to remove extracellular viruses and inactivated with PMSF. Intracellular viruses were quantified. In each test, CS served as a control. Error bars represent the SD (n = 3); * p < 0.05.

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  Figure 6. Analysis of effect of FCS on gene expression in DHAV-1-infected DEF cells. (A) Determination of effect of FCS on RNA replication. The negative strand replicative–intermediate of DHAV-1 was quantified at 6 hpi. (B) Schematic diagram of bicistronic reporter plasmids. (C) Analysis of effect of FCS on DHAV-1 IRES-mediated translation. Left panel, DEF cells were transfected with the bicistronic DNA plasmids. The activity of Fluc and Rluc was measured at 24 h after transfection. Right panel, DEF cells were transfected with in vitro transcribed RNA. The activity of Fluc and Rluc was measured at 17 h after transfection. Error bars represent the SD (n = 3).

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  Figure 7. Analysis of effect of FCS on extracellular viral loads. DEF cells inoculated with C-QYD (A) or C80 (B) were maintained with DMEM and treated with FCS at 37 °C by addition of FCS into the wells at 1 and 2 hpi respectively, and CS was included as a control. The supernatants were collected from three well of 24-well plates, and the viral loads were quantified by RT-qPCR assay. Error bars represent the SD (n = 3); * p < 0.05; ** p < 0.01.