The development of probes was based in analysis of the full haemagglutinin and nucleoprotein gene sequences of all Chinese human isolates of H5N1. Oligonucleotide DNA probes representing conserved gene regions were used. Probes were labeled by digoxigenin-UTP (Roche Diagnostics, Penzberg, Germany) and tested for specificty using human biopsy of gut tissue with in vitro infection of H5N1 virus. Since H5N1 is a negative-stranded RNA virus, sense probes were defined as the probes that detect the viral RNA (negative-stranded), whereas antisense probes detected mRNA and complementary RNA (cRNA), which are both positive-stranded. Briefly, before hybridization, all solutions were prepared with DEPC-treated water. After deparaffinization and rehydration, tissue sections of 5 mm thickness were treated with proteinase K digestion for 30 min. Tissue sections were then incubated with a hybridization cocktail containing 25 mg/mL of probes at 37uC for 16,18 h. All sense and antisense probes were applied separately on consecutive tissue sections. After blocking with normal horse serum (1:100), sections were incubated with alkaline phosphatase-labelled digoxigenin antibody (1:1000, Roche Diagnostics, Penzberg, Germany) for 1 h, and the reaction products were colorised with nitroblue tetrazolium/5-bromo-4choloro-3-indolyl phosphate (Roch Diagnostics, Penzberg, Germany) for 1-1.5 h, and counterstained in 2% nucleic fast red liquid for 1 min. As a positive control, we used human lung and gut biopsy tissues with in-vitro infection of H5N1 virus. Negative controls also included an unrelated antisense probe against the fragment of the heamaglutinin gene of the seasonal influenza virus H3N2 as well as H5N1 in-situ hybridization probes to tissues.