Avian reovirus replication in mononuclear phagocytes in chicken footpad and spleen after footpad inoculation

Abstract

Circulating monocytes and tissue macrophages were suggested to be susceptible to avian reovirus (ARV) infection. To determine if ARV infects and replicates in mononuclear phagocytes (KUL01-positive cells), we infected 3-day-old specific-pathogen-free chickens with ARV strain 2408 by inoculation of the left footpad. The left footpads and spleens were collected for analysis at 1.5 and 2.5 d after inoculation. Replication of ARV in the footpad and spleen was demonstrated by detection of the viral protein σNS using immunohistochemical testing and viral S1 RNA expression by real-time quantitative polymerase chain reaction (qPCR). Furthermore, immunofluorescent double-staining assay of cytocentrifuged cells and cryosections of the footpad and spleen for the viral protein σNS and the surface marker recognized by monoclonal antibody (MAb) KUL01 indicated that KUL01-positive cells costained with MAb H1E1, which recognizes ARV protein σNS. In addition, more ARV S1 RNA was measured by qPCR in the KUL01-positive cell samples prepared from the footpad or spleen 1.5 d after inoculation compared with non-KUL01-positive cell samples. The amounts of ARV S1 RNA in the spleen were significantly lower (P < 0.05) than the amounts in the footpad 1.5 d after inoculation. The results suggest that ARV infects mononuclear phagocytes and then replicates within these cells before migrating to the spleen, where it infects and replicates in KUL01-positive cells.

Figure 1

Immunohistochemical evidence of avian reovirus (ARV) 2408 replication in chicken footpad removed 1.5 d (b, original magnification ×40) and 2.5 d (d, original magnification ×40) after inoculation and stained with monoclonal antibody (MAb) H1E1. Sections from the footpad of control chickens collected at 1.5 and 2.5 d were free of protein σNS positive signals; thus, only those at 2.5 d (a) are represented here. Panels c and e show ×10 magnification of the insets in panels b and d, respectively. Some of the signals are indicated by arrows.

Figure 2

Immunohistochemical evidence of ARV 2408 replication in chicken spleen removed 1.5 d (b, original magnification ×40) and 2.5 d (d, original magnification ×40) after inoculation and stained with MAb H1E1. Sections from the spleen of control chickens collected at 1.5 and 2.5 d were free of protein σNS positive signals; thus, only those at 2.5 d (a) are represented here. Panels c and e show ×5 magnification of the insets in panels b and d, respectively. Some of the signals are indicated by arrows. E — periellipsoid lymphoid sheath; A — periarteriolar lymphoid sheath; ▲ — arteriole; * — vein.

Figure 3

Scores (mean ± standard error) for quantities of protein σNS positive signals 1.5 and 2.5 d after virus inoculation in the left footpad and spleen sections shown in Figures 1 and 2, respectively, as determined by counting the signals in 5 random fields at a magnification of ×400 and averaging the results. 0 — no positive signal; > 0 and < 1 — mild quantities of signals; > 1 and < 2 — moderate quantities of signals; > 2 and < 3 — marked quantities of signals. Two independent experiments were carried out. ** — P < 0.01.

Figure 4

Levels [mean ± standard deviation (SD)] of ARV S1 RNA expression in the footpad and spleen in experiment 1 as measured by real-time quantitative polymerase chain reaction (qPCR) 1.5 and 2.5 d after virus inoculation. All tissue samples from the control birds were free of ARV S1 RNA. Each sample was assessed in triplicate. Two independent experiments were carried out. ** — P < 0.01; * P < 0.05.

Figure 5

Results of immunofluorescent double-staining assay for ARV protein σNS in KUL01-positive cells in cryosections of left footpad (A) and spleen (B) and in cytocentrifuged cells of the left footpad (C) of infected or control chickens 2.5 d after inoculation. Two independent experiments were carried out. Viral σNS stains red, the mononuclear phagocyte marker KUL01 stains green, and nuclei visualized with 4′,6-diamidino-2-phenylindole stain blue. Arrows indicate infected cells. Magnification ×200.

Figure 6

Levels (mean ± SD) of ARV S1 RNA expression in KUL01- positive cells (darker columns) and non-KUL01-positive cells (lighter columns) from the footpad and spleen as measured by qPCR 1.5 d after virus inoculation. The leukocyte fraction was prepared and stained with fluorescein isothiocyanate (FITC)-conjugated MAb KUL01, and the KUL01- positive cells were purified by positive selection with the use of magnetically labeled anti-FITC antibody and magnetic-activated cell-sorting column separation. Other cell populations in the flow-through fraction were collected and treated as the non-KUL01-positive cells. Total RNA was extracted from both types of samples and used for qPCR. All tissue samples from the controls were free of ARV S1 RNA. Each sample was assessed in triplicate. Two independent experiments were carried out. The data are expressed as means ± SD. ** — P < 0.01; * P < 0.05.