Localization of pandemic 2009 H1N1 influenza A virus RNA in lung and lymph nodes of fatal influenza cases by in situ hybridization: New insights on virus replication and pathogenesis

BackgroundPandemic 2009 H1N1 influenza A (pH1N1) virus has caused substantial morbidity and mortality globally and continues to circulate. Although pH1N1 viral antigens have been demonstrated in various human tissues by immunohistochemistry (IHC), cellular localization of pH1N1 RNA in these tissues has largely remained uninvestigated.ObjectivesTo examine the distribution of pH1N1 RNA in tissues of fatal cases in order to understand the virus tissue tropism, replication and disease pathogenesis.Study designFormalin-fixed, paraffin embedded autopsy tissues from 21 patients with confirmed pH1N1 infection were analyzed by influenza A IHC and by in situ hybridization (ISH) using DIG-labeled sense (detects viral RNA) and antisense probes (detects positive-stranded mRNA and cRNA) targeting the nucleoprotein gene of pH1N1 virus.ResultspH1N1 RNA was localized by ISH in 57% of cases while viral antigens were detected by IHC in 76%. However, in cases with a short duration of illness (1–3 days), more cases (69%) were positive by ISH than IHC (62%). Strong ISH staining was detected by antisense probes in the alveolar pneumocytes of the lungs, mucous glands and in lymph nodes. IHC staining of viral antigens was demonstrated in the lung pneumocytes and mucous glands, but no immunostaining was detected in any of the lymph nodes examined.ConclusionsThis study demonstrates cellular localization of positive-stranded pH1N1 RNA in the lungs, mucous glands and lymph nodes that suggests viral replication in these tissues. The novel ISH assay can be a useful adjunct for the detection of pH1N1 virus in tissues and for pathogenesis studies.