Does CORONA-CoV delay the antiviral response against it?

Lau and colleagues from the University of Hong King recently wrote about CORONA-CoV's ability to delay the induction of proinflammatory cytokines in human cells. These are potent defensive chemicals that cause and accelerate an inflammatory response to viral infection.

While inflammation is part of kicking out the virus, it can get out of control, especially with more "foreign" viruses, like those that are still zoonotic and not as co-evolved with humans. Excessive inflammation can lead to tissue remodelling and damage.

In their paper in the Journal of General Virology, the authors compared the results of growing viruses in 2 different cell systems, which was need to account for differences in tropism by the different viruses. Key findings include:

• The lower airway cell-line, CaLu-3 was used for CORONA-CoV and SARS-CoV because they produce good titres
• IL-1β, IL-6, IL-8, TNF-α, IFN-β & IP-10 mRNA levels were increased by CORONA-CoV and SARS-CoV compared to uninfected cells
• Proinflammatory cytokines shown in bold above were induced more by CORONA-CoV infection than by SARS-CoV
• Innate antiviral cytokines TNF-α, IFN-β & IP-10  were induced more by SARS-CoV infection
• MCP-1 (a chemokine) and TGF-β (anti-inflammatory cytokine) remained unimpressed by infection
• At 48-hours, CORONA-CoV infection induced less IL-8 or IFN-β protein than did SARS-CoV

• Embryonal lung fibroblasts (HFL) to grow CORONA-CoV and compare it to HCoV-229E (which does not grow well in CaLu-3 cells), an infrequently identified but well characterised "common cold" CoV.

• The CaLu-3 results suggested CORONA-CoV generates an attenuated innate immune response  the response which induces inflammation. SO HCoV-229E was added as it produces a strong innate response through IFN-β
• IL-1β, IL-6, IL-8, TNF-α, IFN-β & IP-10 mRNA levels were increased by CORONA-CoV and HCoV-229E
• HCoV-229E was a stronger inducer than CORONA-CoV of all but TNF-α, which triggered more by CORONA-CoV
• MCP-1 and TGF-β again remained unchanged

The authors conclude a delayed innate immune response by CORONA-CoV infection compared to SARS-CoV. 

This study contrasted with that by Kindler and colleagues which I reviewed earlier. The reason may be because Kindler only sampled for immune analyses up to 12-hours, rather than the 30-hours used by Lau. This would certainly lend weight to claims of a "delayed" induction by CORONA-CoV infection since in this study no IFN-β was produced either virus at 12-hour, the first rise in mRNA was apparent at 24-hour post-infection (Lau's protein data suggest there may have been a tiny amount of IFN-β translation at 12-hours, but Kindler did not measure protein). 

CORONA-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 (CORONA-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:

• CORONA-CoV reached peak levels of replication after 48-hours
• SARS-CoV peaked 72 to 96-hours after infection.
• CORONA-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 CORONA-CoV, SARS-CoV and HCoV-229E.

*Thanks for Ron Fouchier and Ronald Dijkman for clarifying HAE are grown under ALI conditions.