Localization of Avian Influenza Virus in Formalin-Fixed, Paraffin-Embedded Chicken Tissues by In Situ Hybridization

In this study, in situ hybridization (ISH) was developed to detect avian influenza virus (AIV) in Madin-Darby canine kidney (MDCK) cells and formalin-fixed, paraffin-embedded chicken tissues. A cDNA probe corresponding to a region of AIV nucleoprotein (NP) gene was synthesized and labeled with digoxigenin. Probe specificity was determined by AIV infected MDCK cells in vitro and the results showed that strong cytoplasmic staining was only detected in AIV-infected cells. Various tissues were collected from 12 h to 35 days post-infection (PI) following inoculation with the H9N2 subtype AIV. AIV was localized in the epithelial cells of the duodenum and cartilage of the throat and trachea at 12 h PI. Tissues from uninfected chickens were negative. The finding of this study indicated ISH was a sensitive and specific technique to detect and localize AIV as well as to study AIV pathogenesis.