Friday, 11 October 2013

MERS-CoV, SARS-CoV, HCoV-229E: comparative culture and immunology

The following study in mBio by Kindler and colleagues from Switzerland, Germany, Denmark and the Netherlands came out in February but, as I'm trying to brush up for a talk next week, it's an important one to add to my recent list of quick reviews.

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.