Figure 1.

In-situ hybridisation locating gene sequences of H5N1 viral antigens (nucleoprotein and haemagglutinin)

Signals seen with nitroblue tetrazolium/5-bromo-4-choloro-3-indolyl phosphate (purple-blue) and immunohistochemical signals with 3-amino-9-ethylcarbazole (double labelling, brown-red). No counterstaining done unless stated otherwise. (A) Lung tissue showing severe damage, hyaline membrane formation, and oedema (by haematoxylin and eosin staining), by contrast with limited number of cells positive for in-situ hybridisation in lung tissue of patient 1 (figure 1B). (B) Positive signals (with nucleoprotein sense probe) in nuclei of isolated pneumocytes (arrows). (C) Double labelling of in-situ hybridisation (with nucleoprotein antisense probe) and immunohistochemistry (with tubulin-β antibody; brown, arrowheads) showing dark-blue viral genomic sequences (arrows) in cytoplasm of tubulin-negative non-ciliated cell (arrow 1) and tubulin-positive ciliated cell (arrow 2) in trachea. No signal seen in nuclei (lightly counterstained with methyl-green). (D) Positive signals (with nucleoprotein sense probe) in several mononuclear cells in lymph node (arrows, lightly counterstained with methyl-green). (E) Positive signals (with nucleoprotein antisense probe) in mucosal epithelial cells of small intestine (arrows, lightly counterstained with methyl-green). (F) Positive signals (with nucleoprotein sense probe) in brain cells (arrows) from left parietal lobe (lightly counterstained with methyl-green), mainly located in cytoplasm and confirmed to be neurons from immunostaining for neurofilament or neuron-specific enolase (webfigures 1O and 1P). (G) Positive signals (with nucleoprotein antisense probe) in large mononuclear cells with morphological features of cytotrophoblasts in periphery of chorionic villus (arrows). Cells confirmed as cytotrophoblasts (webfigure 4D). No positive signals noted in any syncytiotrophoblasts. (H) Positive signals (with nucleoprotein sense probe) in fetal liver cells (arrows), confirmed as Kupffer cells (webfigure 1F). (I) Positive signals (with haemagglutinin sense probe) in bronchiolar epithelial cells in fetal lung tissue (arrows).