HEV infection in intravenously inoculated wild boar and in dexamethasone-treated

Biochemical parameters and serology

At the end of experiment elevated ALT and GGT levels in serum were observed in groups 1A and 1B at 28 to 30 dpi (Figure 4.1 A). Other biochemical parameters remained within normal limits. Anti-HEV antibodies were detected first time in group 1A at 6 dpi and in group 1B at 9

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dpi. In comparison to group 1A, mean OD450-values in group 1B were slightly increased at 28 to 30 dpi (Figure 4.1 B). In each group, 3 out of 4 wild boar had detectable antibodies at 13 dpi, but only 1 out of 4 in group 1A and 2 out of 4 animals in group 1B were still tested positive at 30 dpi (Table 4.2). No significant influence of the immunosuppressive status (without versus with dexamethasone treatment) on liver enzyme levels and the antibody response to HEV inoculation was provable, but ALT and GGT levels in group 1A were significantly increased compared to the reference value range (Figure 4.1).

Detection of HEV RNA

Viral RNA in serum was only found transiently in one wild boar group at 21 dpi (group 1B) respectively at 28 dpi (group 1A), and in both at 30 dpi (Table 4.2). In fecal samples HEV RNA was detected at first in group 1A at 4 dpi and in group 1B at 6 dpi. All wild boar in group 1A shed viral RNA in feces within 28 to 30 dpi, whereas fecal shedding was detectable in all animals of group 1B already at 21 dpi and lasted until the end of experiment (Table 4.3).

The time course of HEV RNA detection in serum and feces of wild boar was summarized in Figure 4.1 C. Viral RNA was also detected in bile, small and large intestine, cecal ingesta, liver and gall bladder of all wild boar, whereas in both groups 3 out of 4 liver lymph nodes, in group 1A 3 out of 4 and in group 1B 2 out of 4 spleen samples were tested positive. An overview of the mean HEV excretion including the proportion of positive tested specimens is given in Figure 4.2 A, and the mean viral load in different tissues with the number of positive tested tissue samples is shown in Figure 4.2 B. HEV RNAs were also found in other lymphatic tissues and different intestinal locations of the wild boar (Additional file 3). No significant differences concerning HEV RNA excretion and viral load in tested tissue samples were present between group 1A and 1B (Figure 4.2).

Liver histopathology and distribution of viral antigens in different tissues

Mild lymphoplasmacytic infiltrates with single cell necrosis of hepatocytes and Kupffer cell proliferations were found in the livers of all intravenously inoculated wild boar. Partially, hepatic lesions were associated with mild infiltrates of CD3 positive cells, but without marked differences between group 1A and 1B. By IHC, several tissue samples were analyzed for the distribution of viral antigens (Figure 4.8). Detection of viral antigens in tissue samples of wild

boar was not consistent with group allocation. In group 1A and 1B viral antigens were found in liver (3/8), liver lymph nodes (4/7), spleen (1/8) and mesenteric lymph node (1/8). All IHC results of viral antigen detection are summarized in the Additional file 4.

Cellular immune responses

Hematological analysis (Figure 4.3) revealed a transient decrease of WBC between 4 and 6 dpi in both wild boar groups which was significant in group 1A compared to the reference value range. In both groups the decrease was followed by an increase of WBC after 13 dpi.

WBC counts were much more pronounced in group 1A and significantly distinct between both groups at 13 dpi. Similarly, a slight decrease of LYM at 4 dpi was followed by an increase after 13 dpi in both groups. Lymphocytosis persisted until 30 dpi and LYM counts were significantly distinct between both groups at 13, 21, 28 and 30 dpi. A slight monocytosis was seen in group 1A at 13 to 15 dpi and in group 1B at 9 to 15 dpi, respectively at 21 to 24 dpi. At 4 to 9 dpi a decrease of NEU was observed in both groups followed by an increase in group 1A at 15 dpi.

Parameters indicative for the T cell populations in peripheral blood are summarized in Figure 4.4: All inoculated animals showed a marked elevation of the absolute number of cytotoxic T lymphocytes (CD8+CD4-) starting from 6 dpi to 30 dpi. This increase was significant in group 1A compared to 0 dpi and significantly distinct between both groups at 30 dpi. Only slight changes were observed in both groups. Following the increase of cytotoxic T lymphocytes (CD8+CD4-), an increase of T helper/memory cells (CD4+CD8+) was detectable starting at 15 dpi. This increase was significant in group 1A compared to 0 dpi and significantly distinct between both groups at 30 dpi. The percentage of activated γδ T cells (γδTCR+CD8+) increased in all animals after 13 dpi with a peak level at 18 dpi. In the numbers of γδ T cells (γδTCR+), T helper cells (CD4+CD8-) and activated T helper cells (CD4+CD25high) only slight changes were seen in peripheral blood (data not shown). With regard to the B cell population in peripheral blood (Figure 4.5) the percentage of naïve B cells (CD2+CD21+) and of B cells after activation (CD2-CD21+) declined initially in all inoculated wild boar. Cells representing the phenotype of antibody-forming and/or memory B cells (CD2+CD21-) showed a percentage increase in all inoculated groups with highest changes after 13 to 30 dpi (significant differences in group 1A compared to 0 dpi).

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Immune cell phenotypes of separated cells of liver, spleen and mesenterial lymph node were examined after necropsy at 30 dpi (Figure 4.6). Dexamethasone controls (group 1C) were included into the analyses as a comparison group. Lower percentages of T-cells were observed in liver of group 1A and 1B with significant differences between both groups.

Moreover, decreased percentages of cytotoxic T lymphocytes (CD8+CD4-), T helper cells (CD4+CD8-) and T helper/memory cells (CD4+CD8+) were observed in group 1A and 1B.

Conversely, marked increase in percentages of γδ T cells (γδTCR+) compared to group 1C was seen, but no influence of dexamethasone treatment. In spleen, T cells in group 1B were decreased compared to group 1A and 1C, and an influence of dexamethasone treatment in group 1A and 1B was provable. In contrast to the liver, lower percentages of γδ T cells (γδTCR+) compared to group 1C were seen in spleen, but no influence of dexamethasone treatment. Cytotoxic T lymphocyte (CD8+CD4-) and T-helper/memory cell (CD4+CD8+) percentages were slightly reduced in group 1A and 1B, whereas the percentage of T-helper cells was significantly increased compared to group 1C. No remarkable changes in T cell populations were observed in the mesenterial lymph node, except one significant difference in the percentages of T-helper/memory cells (CD4+CD8+) between group 1B and 1C.

4.4.2 HEV transmission to non-treated domestic pigs and dexamethasone-treated