Neutrophils may be a vehicle for viral replication and dissemination in human H5N1 avian influenza

Abstract

The mechanism of systemic spread of H5N1 virus in patients with avian influenza is unknown. Here, H5N1 nucleoprotein and hemagglutinin were identified by immunohistochemistry in the nucleus and cytoplasm of neutrophils in the placental blood of a pregnant woman. Viral RNA was detected in neutrophils by in situ hybridization and enhanced real-time polymerase chain reaction. Therefore, neutrophils may serve as a vehicle for viral replication and transportation in avian influenza.

Figure 1

Nucleoprotein (NP) and hemagglutinin detected in neutrophils. A, Hematoxylin and eosin staining after destaining of the positive immunostaining response performed to identify neutrophils (black arrows; bar, 20 µm). B, Immunohistochemistry with antibody against NP (1:300; Abcam) performed to identify the viral proteins in the nuclei of neutrophils (brown-red; black arrows; bar, 20 µm). C, CD15 (brown-red, black arrow)also present in the NP-positive cells (purple-blue, black arrow; bar, 50 µm). Inset b shows enlargement of a cell highlighted by an arrow in inset a, clearly indicating colocalization of NP (viral protein) and CD15 (neutrophil marker) in the same cells (bar, 10 µm).

Figure 2

H5N1 nucleotide sequences detected in neutrophils by in situ hybridization, microdissection, and enhanced real-time PCR (ERT-PCR). A, Positive in situ hybridization signals of H5N1 (purple-blue; black arrows) detected in CD15-positive cells (red-brown; black arrows) in placental villi (inset a). Macrophages and trophoblastic cells in placental villi were also positive (purple-blue; red arrows; bar, 50 µm). Inset b shows enlargement of inset a, which shows that the same neutrophils contain H5N1 nucleotide sequences (bar, 10 µm). B and C, Three thousand leukocytes, dissected from the infected placenta as the test group. B, Before laser microdissection (bar, 50µm). C, After laser microdissection (bar, 50µm). D and E, Blood preparations with no leukocytes, dissected from the infected placenta as the blank group. D, Before laser microdissection (bar, 50 µm). E, After laser microdissection (bar, 50 µm). Three thousand leukocytes from uninfected placenta were also dissected as a negative control (data not shown). F, Measurement of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) by reamplification of PCR. Lane 1, GAPDH PCR of the positive control group. PCR reamplification of GAPDH of the positive control group (lane 2), on the test group (lane 3), of the negative control group (lane 4), and of the blank group (lane 5). Lane 6, GAPDH PCR of water. Lane 7, PCR reamplification of GAPDH of water. G, Measurements of H5 ERT-PCR for different samples. 1, ERT-PCR measurement of the H5-positive control group. 2, Real-time PCR measurement of the H5-positive control group. ERT-PCR measurement of the test group (3), of the blank group (4), and of the negative control group (5). 6, Real-time PCR measurement of water. 7, ERT-PCR measurement of water. bp, Base pairs.