title: Culturing of respiratory viruses in well-differentiated pseudostratified human airway epithelium as a tool to detect unknown viruses.
abstract: BACKGROUND
  Currently, virus discovery is mainly based on molecular techniques. Here, we propose a method that relies on virus culturing combined with state-of-the-art sequencing techniques. The most natural ex vivo culture system was used to enable replication of respiratory viruses.
  
  
  METHODS
  Three respiratory clinical samples were tested on well-differentiated pseudostratified tracheobronchial human airway epithelial (HAE) cultures grown at an air-liquid interface, which resemble the airway epithelium. Cells were stained with convalescent serum of the patients to identify infected cells and apical washes were analyzed by VIDISCA-454, a next-generation sequencing virus discovery technique.
  
  
  RESULTS
  Infected cells were observed for all three samples. Sequencing subsequently indicated that the cells were infected by either human coronavirus OC43, influenzavirus B, or influenzavirus A. The sequence reads covered a large part of the genome (52%, 82%, and 57%, respectively).
  
  
  CONCLUSIONS
  We present here a new method for virus discovery that requires a virus culture on primary cells and an antibody detection. The virus in the harvest can be used to characterize the viral genome sequence and cell tropism, but also provides progeny virus to initiate experiments to fulfill the Koch's postulates.
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Seyed Mohammad Jazaeri
authors.initials: SM
authors.lastname: Farsani
authors.forename: Martin
authors.initials: M
authors.lastname: Deijs
authors.forename: Ronald
authors.initials: R
authors.lastname: Dijkman
authors.forename: Richard
authors.initials: R
authors.lastname: Molenkamp
authors.forename: Rienk E
authors.initials: RE
authors.lastname: Jeeninga
authors.forename: Margareta
authors.initials: M
authors.lastname: Ieven
authors.forename: Herman
authors.initials: H
authors.lastname: Goossens
authors.forename: Lia
authors.initials: L
authors.lastname: van der Hoek
mesh_terms: D017934:Coronavirus / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification*; D003588:Cytopathogenic Effect, Viral; D004847:Epithelial Cells / Q000166:cytology / Q000276:immunology / Q000821:virology*; D016679:Genome, Viral; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D009981:Influenza B virus / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D008875:Middle Aged; D009975:Orthomyxoviridae / Q000235:genetics / Q000254:growth & development* / Q000276:immunology / Q000302:isolation & purification*; D061251:Primary Cell Culture; D020545:Respiratory Mucosa / Q000166:cytology / Q000821:virology*; D014776:Virus Cultivation / Q000379:methods*; D014779:Virus Replication
keywords: Airway epithelial cultures; VIDISCA-454; influenzavirus B; respiratory viruses; virus discovery