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, 9 (1), 51-7

Culturing of Respiratory Viruses in Well-Differentiated Pseudostratified Human Airway Epithelium as a Tool to Detect Unknown Viruses


Culturing of Respiratory Viruses in Well-Differentiated Pseudostratified Human Airway Epithelium as a Tool to Detect Unknown Viruses

Seyed Mohammad Jazaeri Farsani et al. Influenza Other Respir Viruses.


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.


Figure 1
Figure 1
Immunostaining of infected human airway epithelial (HAE) cell cultures with patient sera. Three respiratory samples, S2705 (HCoV-OC43), I2125 (influenzavirus A, H3N2), and E1517 (influenzavirus B), were used to inoculate human airway epithelial (HAE) cell cultures. The cells were fixed 96 or 120 hours post-inoculation with 4% PFA and immunostained with the autologous antibody derived from respective patients (red) and mouse monoclonal anti-ß-tubulin IV (green), and examined by confocal microscopy. The difference in intensity of tubulin staining represents experimental variation in differentiation (S2705 versus E1517) or a difference in focus depth (I2125, below the apical surface). An overlay image was generated to determine the cell tropism of each virus. Bars: 30 μm.
Figure 2
Figure 2
Replication of influenzavirus B in human airway epithelial (HAE) cell culture. Apical harvests of the E1517 culture were collected at 2, 24, 48, 72, and 96 hours post-inoculation. Viral RNA was quantified by real-time PCR as previously described.

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