4. MANUSCRIPT II: IMMUNE RESPONSE AND VIRAL REPLICATION IN
4.7 Figures
Figure 4.1 Mean values of liver enzyme levels, antibody responses and HEV RNA loads in serum and feces of wild boar.
Error bars represent ±SD. A. Detection of alanine aminotransferase (ALT) and gamma-glutamyl transferase (GGT) in serum of group 1A, 1B and 1C. Upper reference range limit = grey-dotted line. IU = international units. Two way ANOVA (Bonferroni t-test; P<0.05). The difference in the mean values among group 1A and the uninfected reference group “0 DPI” is greater than would be expected by chance: *c = significant with P<0.05.
B. Detection of anti-HEV IgG-antibodies in serum of group 1A and 1B with mean absorbance (OD450).
PrioCHECK® HEV Ab porcine. Cut-off (grey-dashed line): OD450 > 0.6. One way ANOVA (Bonferroni t-test;
P<0.05). C. HEV RNA in serum and feces of group 1A and 1B estimated by RT-qPCR. One way ANOVA (Bonferroni t-test; P<0.05). DPI = day post inoculation of group 1A and 1B.
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Figure 4.2 Viral loads in different excreta and tissue samples of wild boar and domestic pigs.
A. Mean excreted HEV genome equivalents in bile, small and large intestine, ingesta (of cecum) and feces with positive standard deviation (only reactive samples considered). The proportion of positive tissue samples is included in each bar column (n positive sample / n total sample). One way ANOVA (Bonferroni t-test; P<0.05);
n.s. = not significant P>0.05. B. Mean HEV genome equivalents in liver, gall bladder, liver lymph node and spleen with positive standard deviation (only reactive samples considered). The proportion of positive tissue samples is included in each bar column (n positive sample / n total sample). One way ANOVA (Bonferroni t-test; P<0.05); n.s. = not significant P>0.05. DPI = day post inoculation of group 1A and 1B.
Figure 4.3 Differential cell counts in peripheral blood of wild boar.
The mean number (in K/µL) of leukocytes (WBC), lymphocytes (LYM), monocytes (MON) and neutrophils (NEU) were determined using an automated XT-2000iV hematology analyzer. Error bars represent ±SD.
Reference value ranges (grey-dotted line) for the tested blood parameters in wild boar were calculated for the evaluation of the results. Two way ANOVA (Bonferroni t-test; P<0.05). The difference in the mean values among group 1A and 1B is greater than would be expected by chance: *a = significant with P<0.05. The difference in the mean values among group1C and the uninfected reference group “0 DPI” is greater than would be expected by chance: *b = significant with P<0.05. The difference in the mean values among group1A and the uninfected reference group “0 DPI” is greater than would be expected by chance: *c = significant with P<0.05.
DPI = day post inoculation of group 1A and 1B.
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Figure 4.4 T-cell related responses upon infection in blood lymphocytes of wild boar.
The number of T cell subpopulations of blood lymphocytes is given: cytotoxic T lymphocytes (CD8+CD4-), T helper/memory cells (CD4+CD8+) and activated γδ T cells (γδTCR+CD8+). Two way ANOVA (Bonferroni t-test; P<0.05). The difference in the mean values among group 1A and 1B is greater than would be expected by chance: *a = significant with P<0.05. The difference in the mean values among group 1C and the uninfected reference group “0 DPI” is greater than would be expected by chance: *b = significant with P<0.05. The difference in the mean values among group 1A and the uninfected reference group “0 DPI” is greater than would be expected by chance: *c = significant with P<0.05. DPI = day post inoculation of group 1A and 1B.
Figure 4.5 B-cell related responses upon infection in blood lymphocytes of wild boar.
Blood lymphocytes were immune-stained to determine the frequency of different B cell subpopulations by FACS analysis: naïve B cells (CD2+CD21+), activated B cells upon antigen contact (CD2-CD21+) and antibody-forming and/or memory B cells (CD2+CD21-). Two way ANOVA (Bonferroni t-test; P<0.05). The difference in the mean values among group 1C and the uninfected reference group “0 DPI” is greater than would be expected by chance: *b = significant with P<0.05. The difference in the mean values among group 1A and the uninfected reference group “0 DPI” is greater than would be expected by chance: *c = significant with P<0.05.
DPI = day post inoculation of group 1A and 1B.
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Figure 4.6 T-cell related responses upon infection in liver, spleen and mesenterial lymph node of wild boar.
Immune cell phenotypes of separated cells of liver, spleen and mesenterial lymph node were examined after necropsy: T cells (CD3+), cytotoxic T lymphocytes (CD8+CD4-), T helper cells (CD4+CD8-), T helper/memory cells (CD4+CD8+) and γδ T cells (γδTCR+). A. T cell related responses in wild boar. Two way ANOVA (Bonferroni t-test; P<0.05). The difference in the mean values among group 1A and 1B is greater than would be expected by chance: *AB = significant with P<0.05. The difference in the mean values among group 1A and 1C is greater than would be expected by chance: *AC = significant with P<0.05. The difference in the mean values among group 1B and 1C is greater than would be expected by chance: *BC = significant with P<0.05. The difference in the mean values among group 1A, 1B and 1C is greater than would be expected by chance: *ABC
= significant with P<0.05.
Figure 4.7 T-cell related responses upon infection in liver, spleen and mesenterial lymph node of domestic pigs.
Immune cell phenotypes of separated cells of liver, spleen and mesenterial lymph node were examined after necropsy: T cells (CD3+), cytotoxic T lymphocytes (CD8+CD4-), T helper cells (CD4+CD8-), T helper/memory cells (CD4+CD8+) and γδ T cells (γδTCR+). A. T cell related responses in wild boar. Randomly selected HEV negative domestic pigs (n = 6) served as negative controls. Two way ANOVA (Bonferroni t-test; P<0.05). The difference in the mean values among group 2A and 2B is greater than would be expected by chance: *AB = significant with P<0.05. The difference in the mean values among group 2A and 2C is greater than would be expected by chance: *AC = significant with P<0.05. The difference in the mean values among group 2B and control is greater than would be expected by chance: *BC = significant with P<0.05. The difference in the mean values among group 2A, 2B and control is greater than would be expected by chance: *ABC = significant with P<0.05. Control = negative controls.
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Figure 4.8 Detection of viral antigens in the liver, liver lymph node and spleen of wild boar by immunohistochemistry.
A - C: Diffuse distribution of viral antigens within a liver lobule (group 1B). D - E: Viral antigens in the cortical zone (central area of the node) and in secondary follicles of a liver lymph node (group 1A). F: Immunolabelling of viral antigens in the germinal center of a lymphoid follicle in spleen (group 1B). All scale bars represent 100 µm.