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.
【저자키워드】 respiratory viruses, virus discovery, Airway epithelial cultures, influenzavirus B, VIDISCA-454, 【초록키워드】 Sequencing, Genome, virus, Replication, Antibody detection, Culture, Next-generation sequencing, cell tropism, experiment, molecular, human coronavirus OC43, convalescent serum, airway epithelium, primary cell, Ex vivo, HAE, viral genome sequence, sequence, infected cell, progeny, air–liquid interface, Cell, clinical sample, Result, tested, analyzed, identify, was used, were infected, indicated, the patient, can be used, provide, human airway epithelial, tracheobronchial, were stained, 【제목키워드】 virus, respiratory virus, Human airway epithelium, detect,