Pathological study of archival lung tissues from five fatal cases of avian H5N1 influenza in Vietnam

Highly pathogenic avian H5N1 influenza virus (H5N1) infection in humans causes acute respiratory distress syndrome, leading to multiple organ failure. Five fatal cases of H5N1 infection in Vietnam were analyzed pathologically to reveal virus distribution, and local proinflammatory cytokine and chemokine expression profiles in formalin-fixed, paraffin-embedded lung tissues. Our main histopathological findings showed diffuse alveolar damage in the lungs. The infiltration of myeloperoxidase-positive and/or CD68 (clone KP-1)-positive neutrophils and monocytes/macrophages was remarkable in the alveolar septa and alveolar spaces. Immunohistochemistry revealed that H5N1 mainly infected alveolar epithelial cells and monocytes/macrophages in lungs. H5N1 replication was confirmed by detecting H5N1 mRNA in epithelial cells using in situ hybridization. Quantitation of H5N1 RNA using quantitative reverse transcription PCR assays revealed that the level of H5N1 RNA was increased in cases during early phases of the disease. We quantified the expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-8, regulated on activation normal T-cell expressed and secreted (commonly known as RANTES), and interferon-gamma-inducible protein of 10 kDa (IP-10) in formalin-fixed, paraffin-embedded lung sections. Their expression levels correlated with H5N1 RNA copy numbers detected in the same lung region. Double immunofluorescence staining revealed that TNF-α, IL-6, IL-8 and IP-10 were expressed in epithelial cells and/or monocytes/macrophages. In particular, IL-6 was also expressed in endothelial cells. The dissemination of H5N1 beyond respiratory organs was not confirmed in two cases examined in this study.