Advances for the Hepatitis A Virus Antigen Production Using a Virus Strain With Codon Frequency Optimization Adjustments in Specific Locations

Figures

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  FIGURE 1. Comparative activity of L0 and HP IRES-types in MRC-5, Vero, and FRhK-4 cell lines. In the box plot diagrams, the box represents the 25th, 50th, and 75th percentiles, the whiskers the 5th and 95th percentiles, and the red dashed line the mean. (A) Activity of L0 and HP IRES-types in each cell line. *Significant statistical differences (p < 0.001, p = 0.002 and p < 0.001, in MRC-5, Vero, and FRhK-4 cells, respectively), assessed by the Student’s t-test. (B) Comparative activity of L0 IRES-type and HP IRES-type in each of the three cell lines. *Significant statistical differences (p < 0.001) assessed by the One-way ANOVA test.

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  FIGURE 2. Translation efficiency of a fragment of the VP1 gene, whose codon composition is different in the L0 and HP strains, under the control of L0 and HP IRES-types and in the absence or presence of actinomycin D (AMD) in FRhK-4 cells. In the box plot diagrams, the box represents the 25th, 50th, and 75th percentiles, the whiskers the 5th and 95th percentiles, and the red dashed line the mean. (A) Translation efficiency of the VP1 fragment from the L0 strain. *Significant statistical differences (p < 0.001) assessed by the Student’s t-test. (B) Translation efficiency of the VP1 fragment from the HP strain. *Significant statistical differences (p < 0.001) assessed by the Student’s t-test.

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  FIGURE 3. Growth curves of L0 (red) and HP (green) strains in MRC-5, FRhK-4 and Vero cells.

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  FIGURE 4. Effect of wortmannin on virus release into supernatants of FRhK-4 cells. (A) Virus titers of L0 and HP strains in the absence and presence of wortmannin (W). (B) Fold increase in virus titers in the supernatants with wortmannin treatment.

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  FIGURE 5. Sucrose-Iodixanol gradients of viruses present in the supernatant of FRhK-4 cells infected with the L0 and HP strains in the presence and absence of wortmannin (W). Naked and quasi-enveloped particles have densities of 1.20–1.25 and 1.06–1.12 g/cm3, respectively. The highest number of genome copies of quasi-enveloped particles in the absence or presence of wortmannin are depicted by dashed pink and cyan lines, respectively.

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  FIGURE 6. Antigen detection in supernatants of FRhK-4 infected cells in the absence or presence of wortmannin (W). (A) Mean percentage of replicas positive by an ELISA detecting the HAV antigen. (B) Mean of ELISA units present in the positive samples.

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  FIGURE 7. Growth curves of L0 (red) and HP (green) strains in FRhK-4-BK cells at different MOIs.