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)-� resulted from infection by any CoV
• Only marginal expression of proinflammatory cytokines (TNF-a 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-a (a type I IFN) or IFN-?3 (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-a or IFN-?3 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.

Read More

Combined interferon and ribavirin therapy an option for early MERS-CoV intervention?

Falzarano and colleagues write in Nature Medicine that the combination of ribavirin (an antiviral) and interferon-a2b (a cytokine key to our antiviral defenses) improved the health of MERS-CoV (EMC/2012) inoculated rhesus macaque monkeys. 

This builds on the March Study by Falzarano et al, which reported  the usefulness of this drug cocktail in reducing virus production in a cell culture system.

Treated infected animals did not suffer from increased respiratory rate, breathing difficulties or increased white blood cell counts (mostly due to neutrophils). Treated infected animals also showed no X-Ray changes in their lungs 1-day after infection and little change beyond that. Untreated animals showed mild to marked evidence of pneumonia and also lower levels of markers of inflammation and copies of viral RNA genome.

The macaques were inoculated with 10⁶ tissue culture infective doses (TCID₅₀) by combined intratracheal (lower respiratory tract), intranasal (upper respiratory tract), oral (ingestion) and ocular routes. That seems to represent a fairly "shock-and-awe" cocktail of inoculation routes compared to how humans may get exposed. Viral RNA was detected in spleen, kidney, lymph nodes, upper and lower respiratory tract and airways in the untreated animals - and many of those sites in the treated ones. The relevance of this study to MERS-CoV transmission is probably limited, but then it's not a transmission study, it's a treatment study. There is another article cited as in press from part of this group. It may further address tissue replication; Novel human betacoronavirus causes a transient lower respiratory tract infection in a rhesus macaque model. Proc. Natl. Acad. Sci. USA.

Interestingly, disease in the macaques is at best mild to moderate in severity, so how the drug cocktail would work when faced with severe human disease...which may take longer to develop...remains unclear. Also unclear, is what disease would look like in macaques with a similar degree of comorbidities to those seen in the most severe human MERS cases.

The main proviso is, the cocktail must be administered early; it was administered 8-hours after infecting the monkeys here. 

With a 5-working day to 2-week turnaround in testing for MERS-CoV, treatment would have to be started at first suspicion and even then might not meet this window. Given the Qatar GP story yesterday, that window would be long shut, painted over and boarded up.

Nonetheless, this is a very encouraging finding and a very nice piece of work that adds an important step in the path to providing a therapeutic option to MERS-CoV infections.

Read More

Stuff from the literature: Dabbling ducks respond to flu viruses.

Yes, ducks do mount an immune response even to "low path" influenza virus infections. Dabbling ducks (those that feed near the surface rather than diving underwater) are the natural reservoir for Low Pathogenic Avian Influenza (LPAI).

LPAI viruses circulate among wild birds, especially Mallards, all the time. As Jourdain and colleagues pointed out in 2010, mallards are not obviously affected by experimental infection - for example they don't lose weight or cease moving around and they don't show any other clear signs of disease (very slight temperature rise for 2-days) after infection. However, the authors note other studies identifying egg production problems and also the importance of further studies on wild populations as opposed to a small number of birds.

Perkins and colleagues showed that ducks, sparrows and gulls tend to control virus replication differently from chickens suggesting that the ducks may have a better ability to mount an immune response. A weaker (less antibody and shedding)reinfection with the same H7N7 virus was still possible even in the presence of an antibody response to the first H7N7 infection. Reinfection by a different virus subtype (H5N2) seems to have been blocked by the antibodies made to initial infection by H7N7; so-called heterotypic immunity (cross-protective immunity to a different viral subtype).

Volmer and colleagues reported in 2011 that ducks infected with an LPAI mount an interferon (IFN) response in their guts. The cells (enterocytes) of the duck gut is where most flu virus replication occurs and its from here that most of the virus shedding originates. This study found the gut was inflamed and that there was some cell death. So, not at all like the respiratory disease the human host experiences. In the cells lining the intestine, the authors found lots of myxovirus-resistant (Mx) gene activity; diverse genes in birds, which may be especially well adapted in mallards to control influenza, that are key to the earliest immune response to virus infections (also important in humans) and particularly effective against influenza virus. Mx proteins trap and redirect flu virus nucleoproteins.

In particular, a Type I (antiviral) IFN response was detected. IFN-g (gamma) gene activity was up, activating important immune cells (T cells or natural killer cells), important for flu vaccines in poultry and humans.

Swayne provides an excellent review of the vaccines used in birds to moderate H5N1 disease ('fowl plague'). The formulation, use and effect of these vaccines are as varied as the influenza viruses themselves. China leads the field in the use of reverse genetics to create the contemporary flu viruses carrying the most relevant immune stimulating (antigenic) bits.

Read More