title: 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.
abstract: BACKGROUND
  Pandemic 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.
  
  
  OBJECTIVE
  To examine the distribution of pH1N1 RNA in tissues of fatal cases in order to understand the virus tissue tropism, replication and disease pathogenesis.
  
  
  METHODS
  Formalin-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.
  
  
  RESULTS
  pH1N1 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.
  
  
  CONCLUSIONS
  This 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.
authors.affiliation: Infectious Diseases Pathology Branch, Division of High-Consequence Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA. JBhatnagar@cdc.gov
authors.forename: Julu
authors.initials: J
authors.lastname: Bhatnagar
authors.forename: Tara
authors.initials: T
authors.lastname: Jones
authors.forename: Dianna M
authors.initials: DM
authors.lastname: Blau
authors.forename: Wun-Ju
authors.initials: WJ
authors.lastname: Shieh
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Paddock
authors.forename: Clifton
authors.initials: C
authors.lastname: Drew
authors.forename: Amy M
authors.initials: AM
authors.lastname: Denison
authors.forename: Dominique C
authors.initials: DC
authors.lastname: Rollin
authors.forename: Mitesh
authors.initials: M
authors.lastname: Patel
authors.forename: Sherif R
authors.initials: SR
authors.lastname: Zaki
mesh_terms: D000293:Adolescent; D000328:Adult; D002648:Child; D002675:Child, Preschool; D005260:Female; D006801:Humans; D017403:In Situ Hybridization; D007223:Infant; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics* / Q000502:physiology; D007251:Influenza, Human / Q000473:pathology* / Q000821:virology; D008168:Lung / Q000473:pathology* / Q000821:virology; D008198:Lymph Nodes / Q000473:pathology* / Q000821:virology; D008297:Male; D008875:Middle Aged; D012367:RNA, Viral / Q000032:analysis*; D014779:Virus Replication; D055815:Young Adult
chemical_list: D012367:RNA, Viral