Comparative analysis of virus-host interactions caused by a virulent and an attenuated duck hepatitis A virus genotype 1

Fig 1. Codon usage bias analysis and fixed SNPs in attenuated and virulent strains. A) RSCUs used in virulent and chick embryo attenuated strains were subject to hierarchical cluster analysis according to their Euclidean distances (Hemi software). The results indicated that the RSCU was apparently changed in the process of passage in chick embryos. B) The significance of differences of RSCU between the chick embryo attenuated strains and the virulent strains were analyzed by Student-t test. The ratios (A/V) at 1.0 and 1.1 are displayed as two dash lines. *P < 0.05, **P < 0.01 or ***P < 0.001 indicate the level of statistical significance of the differences between different groups. C) Fixed SNPs in the genomes of virulent strains and chick embryo attenuated strains. The conserved nucleotides used by virulent strains are listed on the first line, which corresponds to the dark spots. Red color indicated synonymous mutations or mutations in UTR. Green color indicated missense mutations, which are indicated at each site.

Fig 2. Expression pattern of PRRs in different organs. The relative expression levels of TLR7, TLR3, RIG-1 and MDA5 were calculated by the 2-ΔΔCt method. The error bars represent standard errors of the mean. Statistically significant differences between the mean gene expression levels in different organs were determined by one-way ANOVA (P < 0.05). *P < 0.05, **P < 0.01 or ***P < 0.001 indicate the level of statistical significance of differences between different groups.

Fig 3. Expression patterns of interferons in different organs. The relative expression level of IFN-α, IFN-β and IFN-γ were calculated using the 2-ΔΔCt method. The error bars represent standard errors of the mean. Statistically significant differences between the mean gene expression levels in different organs were determined by one-way ANOVA (P < 0.05). *P < 0.05, **P < 0.01 or ***P < 0.001 indicate the level of statistical significance of differences between different groups.

Fig 4. Expression patterns of interleukins in different organs. The relative expression levels of IL-1β, IL-2, IL-4 and IL-6 were calculated using the 2-ΔΔCt method. The error bars represent standard errors of the mean. Statistically significant differences between the mean gene expression levels in different organs were determined by one-way ANOVA (P < 0.05). *P < 0.05, **P < 0.01 or ***P < 0.001 indicate the level of statistical significance of differences between different groups.

Fig 5. Expression patterns of MHC and BAFF in different organs. The relative expression levels of MHC-I and MHC-II were calculated using the 2-ΔΔCt method. The error bars represent standard errors of the mean. Statistically significant differences between the mean gene expression levels in different organs were determined by one-way ANOVA (P < 0.05). *P < 0.05, **P < 0.01 or ***P < 0.001 indicate the level of statistical significance of differences between different groups.

Fig 6. Expression patterns of chemokines and β-defensin in different organs. The relative expression levels of CCL19, CCL21 and β-defensin were calculated using the 2-ΔΔCt method. The error bars represent the standard errors of the mean. Statistically significant differences between the mean gene expression levels in different organs were determined by one-way ANOVA (P < 0.05). *P < 0.05, **P < 0.01 or ***P < 0.001 indicate the level of statistical significance of differences between different groups.

Fig 7. Comparative study of virus-host interactions mediated by attenuated and virulent strains in the liver. HE, single staining of the viral capsid, 2A2, 2A3, 3C and 3D and double staining of the viral capsid and CD4+ or CD8+ positive cells were performed to compare the pathological changes, viral protein expression levels and the extent of Th or Tc cell responses caused by the diversity of virulence. The positive staining of the viral capsid, 2A2, 2A3, 3C and 3D was visualized as bluish violet, while the double staining of the viral capsid and CD4+ or CD8+ was identified as red or brown, respectively. The negative control (-) refers to standard HE staining and single and double IHC staining without primary antibody. The relative expression levels of immune-related genes were also calculated using the 2-ΔΔCt method. The error bars represent standard errors of the mean. Statistically significant differences induced by the attenuated and virulent strains between the mean gene expression levels were determined by one-way ANOVA. *P < 0.05, **P < 0.01 or ***P < 0.001 indicate the level of statistical significance of differences between different groups.

Fig 8. Comparative study of virus-host interactions mediated by attenuated and virulent strains in the spleen. HE, single staining of the viral capsid, 2A2, 2A3, 3C and 3D and double staining of the viral capsid and CD4+ or CD8+ positive cells was performed to compare the pathological changes, viral protein expression levels and the Th or Tc cell responses caused by the diversity of virulence. The positive staining of the viral capsid, 2A2, 2A3, 3C and 3D was visualized as bluish violet, while the double staining of the viral capsid and CD4+ or CD8+ was identified as red or brown, respectively. The negative control (-) refers to standard HE staining and single and double IHC staining without primary antibody. The relative expression levels of immune-related genes were calculated using the 2-ΔΔCt method. The error bars represent standard errors of the mean. Statistically significant differences induced by attenuated and virulent strains between the mean gene expression levels were determined by one-way ANOVA. *P < 0.05, **P < 0.01 or ***P < 0.001 indicate the level of statistical significance of differences between the different groups.

Fig 9. Comparative study of virus-host interactions mediated by attenuated and virulent strains in the kidney. HE, single staining of the viral capsid, 2A2, 2A3, 3C and 3D and double staining of the viral capsid and CD4+ or CD8+ positive cells were performed to compare the pathological changes, viral protein expression levels and the Th or Tc cell responses caused by the diversity of virulence. The positive staining of the viral capsid, 2A2, 2A3, 3C and 3D was visualized as bluish violet, while the double staining of the viral capsid and CD4+ or CD8+ was identified as red or brown, respectively. The negative control (-) refers to standard HE staining and single and double IHC staining without primary antibody. The relative expression levels of immune-related genes were calculated using the 2-ΔΔCt method. The error bars represent the standard errors of the mean. Statistically significant differences induced by attenuated and virulent strains between the mean gene expression levels were determined by one-way ANOVA. *P < 0.05, **P < 0.01 or ***P < 0.001 indicate the level of statistical significance of differences between the different groups.

Fig 10. Comparative study of virus-host interactions mediated by attenuated and virulent strains in the lung. HE, single staining of the viral capsid, 2A2, 2A3, 3C and 3D and double staining of the viral capsid and CD4+ or CD8+ positive cells were performed to compare the pathological changes, viral protein expression levels and the Th or Tc cell responses caused by the diversity of virulence. The positive staining of the viral capsid, 2A2, 2A3, 3C and 3D was visualized as bluish violet, while the double staining of the viral capsid and CD4+ or CD8+ was identified as red or brown, respectively. The negative control (-) refers to standard HE staining and single and double IHC staining without primary antibody. The relative expression levels of immune-related genes were calculated using the 2-ΔΔCt method. The error bars represent the standard errors of the mean. Statistically significant differences induced by attenuated and virulent strains between the mean gene expression levels were determined by one-way ANOVA. *P < 0.05, **P < 0.01 or ***P < 0.001 indicate the level of statistical significance of differences between the different groups.

Fig 11. Comparative study of virus-host interactions mediated by attenuated and virulent strains in the Harderian gland. HE, single staining of the viral capsid, 2A2, 2A3, 3C and 3D and double staining of the viral capsid and CD4+ or CD8+ positive cells were performed to compare the pathological changes, viral protein expression levels and the Th or Tc cell responses caused by the diversity of virulence. The positive staining of the viral capsid, 2A2, 2A3, 3C and 3D was visualized as bluish violet, while the double staining of the viral capsid and CD4+ or CD8+ was identified as red or brown, respectively. The negative control (-) refers to standard HE staining and single and double IHC staining without primary antibody. The relative expression levels of immune-related genes were calculated using the 2-ΔΔCt method. The error bars represent the standard errors of the mean. Statistically significant differences induced by attenuated and virulent strains between the mean gene expression levels were determined by one-way ANOVA. *P < 0.05, **P < 0.01 or ***P < 0.001 indicate the level of statistical significance of differences between the different groups.

Fig 12. Comparative study of virus-host interactions mediated by attenuated and virulent strains in the thymus. HE, single staining of the viral capsid, 2A2, 2A3, 3C and 3D and double staining of the viral capsid and CD4+ or CD8+ positive cells were performed to compare the pathological changes, viral protein expression levels and the Th or Tc cell responses caused by the diversity of virulence. The positive staining of the viral capsid, 2A2, 2A3, 3C and 3D was developed as bluish violet, while the double staining of the viral capsid and CD4+ or CD8+ was identified as red or brown, respectively. The negative control (-) refers to standard HE staining and single and double IHC staining without primary antibody. The relative expression levels of immune-related genes were calculated using the 2-ΔΔCt method. The error bars represent the standard errors of the mean. Statistically significant differences induced by attenuated and virulent strains between the mean gene expression levels were determined by one-way ANOVA. *P < 0.05, **P < 0.01 or ***P < 0.001 indicate the level of statistical significance of differences between the different groups.