The authors used human bronchial airway epithelium cultures (HAE), also known as air-liquid interface (ALI)* cultures, to grow and examine the immune responses resulting from growth of, the Middle East respiratory syndrome coronavirus (MERS-CoV), the severe acute respiratory syndrome (SARS) CoV or HCoV-229E.
These cultures start life as scraped/brushed/biopsied primary cells that are then "grown out" in special culture flasks in the presence of the right solution of hormones and chemicals, so that they can mimic a true mature epithelium - multilayered, mucous-producing and with beating cilia and tight cell:cell junctions.
So, at 6-hours post-infection with either virus, RNA was purified and run through a next generation sequencing protocol to attempt assembly of the genome of culture virus. Even after culture and using a genome to assemble against, only 0.006% (1,616/24,053,494) of reads could be ascribed to the virus.
Some key points of the culture findings:
- MERS-CoV reached peak levels of replication after 48-hours
- SARS-CoV peaked 72 to 96-hours after infection.
- MERS-CoV infected mostly non-ciliated cells (supporting other findings)
- No induction of interferon (IFN)-β resulted from infection by any CoV
- Only marginal expression of proinflammatory cytokines (TNF-α most active) resulted, mainly by HCoV-229E infection, at 6-hours, suggesting equivalent adaptation of the virus to growth in HAE cultures and that
- Human bronchial epithelium, in the absence of dendritic and other cells, does not mount a strong innate immune response to the CoVs used.
- Uninfected HAE cultures respond quickly to treatment with IFN-α (a type I IFN) or IFN-λ3 (type III IFN), with upregulated expression of RNA from IFN-stimulated genes (ISGs; Mx1, 2'5'OAS, Stat1, Mda5 and Rig-I)
- Addition of IFN-α or IFN-λ3 to try and "protect" sick cells (by pre-incubating with IFN then infecting them) reduced viral genome replication compared to no treatment for MERS-CoV, SARS-CoV and HCoV-229E.
*Thanks for Ron Fouchier and Ronald Dijkman for clarifying HAE are grown under ALI conditions.