Analysis of the 1918 Spanish Flu Virus

Jeffrey Taubenberger, a medical doctor at the Institute of Defense, investigated the 1918 Spanish flu.
He aimed to characterize the virus and understand its evolution by:
- Retrieving fixed tissues from deceased servicemen archived at the Department of Defense.
- Exhuming bodies buried in permafrost in Alaska who died during the pandemic.
Using molecular biology and PCR amplification, they amplified the gene segments of the 1918 Spanish flu.

Reverse genetics was employed to engineer gene segments into DNA plasmids.
These plasmids were designed to produce both positive and negative sense RNA.
Transfecting these plasmids into a cell culture system allowed for the generation of the 1918 Spanish flu virus.
The plasmids, encoding gene segments of the 1918 Spanish flu, are transfected into cell culture.
Positive and negative sense RNAs are produced, leading to viral protein production from the RNA and creation of the viral genome from negative sense RNA.
These components self-assemble into virions, effectively resurrecting the flu virus in a petri dish.
The supernatant containing the virus can be amplified for further study.

Taubenberger and Terence Tumpey characterized the sialic acid binding properties of the resurrected virus.
They compared viruses from the first and second waves of the 1918 Spanish flu.
The "1918 New York" strain, representing the first wave, could bind to both alpha 2-3 (avian linkage) and alpha 2-6 (human linkage) sialic acids.
The 1918 virus, initially identified in New York from returned servicemen, showed affinity for both alpha 2-3 and alpha 2-6 sialic acid linkages.
This dual specificity suggests a possible origin from birds, with subsequent mutations enabling binding to human receptors.
Evolution in the human host led to increased specificity for alpha 2-6 linkages.
The "1918 South Carolina" strain (second wave) exhibited only alpha 2-6 specificity.
The 1918 virus adapted to its human hosts, increasing its specificity for the alpha 2-6 linkage for efficient infection and transmission.
Changes in hemagglutinin binding site correlated with these changes in specificity.

Humans possess both alpha 2-3 and alpha 2-6 sialic acid linkages, but their distribution varies in the respiratory tract.
The upper respiratory tract has high levels of alpha 2-6 linkages, while the lower airways (bronchioles and alveoli) contain cells with alpha 2-3 linkages.
Avian influenza viruses, with alpha 2-3 specificity, face a barrier in infecting the human upper respiratory tract due to the absence of alpha 2-3 linkages.
Infection by avian influenza requires the virus to reach the lower airways through prolonged or high-concentration exposure.
The initial 1918 infection may have involved bird flu introduction, with subsequent evolution from alpha 2-3 to alpha 2-6 specificity through repeated exposures.

The 1918 Spanish flu caused a significant drop in life expectancy with high mortality in young adults (twenties and thirties).
Resurrecting the virus allowed for in-depth understanding of its pathogenicity.
The 1918 virus grew to very high levels compared to seasonal flu strains.
Experiments using reverse genetics showed that multiple gene segments contributed to the virus' virulence.
In ferrets, the 1918 strains caused rapid death within five days, unlike seasonal flu strains.
Swapping polymerase gene segments from 1918 into other strains showed increased virulence, though not as high as the original 1918 strain.

Gene microarray analysis revealed an overactive inflammatory response in ferrets infected with the 1918 virus.
The 1918 infection induced a sustained, high-level inflammatory response, characterized by a type one interferon signature.
Infection with the 1918 virus resulted in a sustained inflammatory response in the lungs, leading to viral primary pneumonia.
The pathogenicity of the 1918 Spanish flu was due to a combination of high viral titer and sustained inflammation in the lungs.
The overactive inflammatory response, termed a cytokine storm, contributed to the severity of the disease and will be discussed later in the context of COVID-19 immunopathology.