Culturing of respiratory viruses in well-differentiated pseudostratified human airway epithelium as a tool to detect unknown viruses

Influenza Other Respir Viruses. 2015 Jan;9(1):51-7. doi: 10.1111/irv.12297. Epub 2014 Dec 8.


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.

Method: 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).

Conclusion: 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.

Keywords: Airway epithelial cultures; VIDISCA-454; influenzavirus B; respiratory viruses; virus discovery.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Coronavirus / genetics
  • Coronavirus / growth & development*
  • Coronavirus / isolation & purification*
  • Cytopathogenic Effect, Viral
  • Epithelial Cells / cytology
  • Epithelial Cells / immunology
  • Epithelial Cells / virology*
  • Genome, Viral
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Influenza A virus / genetics
  • Influenza A virus / growth & development
  • Influenza A virus / isolation & purification
  • Influenza B virus / genetics
  • Influenza B virus / growth & development
  • Influenza B virus / isolation & purification
  • Middle Aged
  • Orthomyxoviridae / genetics
  • Orthomyxoviridae / growth & development*
  • Orthomyxoviridae / immunology
  • Orthomyxoviridae / isolation & purification*
  • Primary Cell Culture
  • Respiratory Mucosa / cytology
  • Respiratory Mucosa / virology*
  • Virus Cultivation / methods*
  • Virus Replication