In this installment we look at a recent paper in the October 7th issue of Science Magazine by Terrence M. Tumpey and coworkers that examines the properties of the 1918 Spanish Flu virus that killed 50 million people world-wide. This is an important paper because it investigate the last really nasty flu virus that jumped from birds directly to humans. Understanding the properties of this virus will help us defend against the bird flu (H5:N1) that is now spreading across the globe. In this important study, the 1918 H1:N1 virus was reconstituted using reverse genetics to yield an active virus. Before we get into the details, it might be wise for you to go read a background on flu virus available in my microbiology textbook.
Various combinations of the 1918 genome viral genome were combined with other H1:N1 flu strains to determine how virulent the strain is and what specific genes lead to that virulence. I think it will be useful to walk through each experiment to give a strong impression of the extreme virulence of the 1918 strain. In the first experiment, the ability of the reconstituted 1918 virus to form plaques on mammalian cells is assayed in the presence and absence of trypsin. For various reasons, the ability to grow in the presence of typsin has been shown to be a property of viruses that are highly pathogenic During viral maturation, the time period after new virus leaves a host cell, but before it enters the next cell, HA needs to be proteolytically cleaved. Highly pathogenic strains have the property of being readily cleaved, in the absence of trypsin. Here is a summary of the results.
Virus
|
Viral growth
|
|
without trypsin
|
with trypsin
|
Tx/91 (an avian strain)
|
+
|
-
|
1918 HA/NA : Tx/91
|
+
|
+
|
1918 NA : Tx/91
|
+
|
+
|
1918 (2)
|
++
|
++
|
Tx/91
is an contemporary human flu strain H1:N1 and is here used as a control. I am not using numbers for clarity and to respect the copyright of Science magazine. What this means is that the neuraminidase (NA) from the 1918 strain is critical in allowing the
1918 virus to spread rapidly in mammals. It must in some way be facilitating the cleavage of HA during maturation of the virus.
In the second experiment presented in the paper, several combination viruses were constructed to test what about the 1918 virus was lethal. A strain containing only the HA of 1918 was combined with the other 7 genes from the Tx/91 strain (1918 HA: Tx/91). This will tests if HA from 1918 is a major determinate of lethality for the 1918
virus. Also, a strain was constructed containing HA, NA, M, NP and NS from 1918 and the three polymerase genes from Tx91 (1918
HA/NA/M/M/NS: Tx/91). If this strain is lethal, then the polymerase is important in the virulence of the microbe. The analysis showed the following.
Virus
|
% Lethality
|
Days
|
1918 (1)
|
100.00%
|
4.5
|
1918 (2)
|
100.00%
|
4.5
|
1918
HA/NA/M/M/NS: Tx/91
|
100.00%
|
8
|
1918
HA/NA:Tx/91
|
0.00%
|
No deaths
|
1918:
Tx/91 HA
|
0.00%
|
No deaths
|
Note the lethality of the two complete 1918 strains. These strains a separate reverse genetic experiments that create the 1918 virus. Every mouse infected with these strains died. Also note that the polymerase genes from the flu virus are not needed to cause lethality as shown by the lethality of the 1918 HA/NA/M/M/NS: Tx/91 strain. Replacement of the HA gene alone in the 1918 virus caused no deaths, indicating the importance of HA in the pathology of the virus. Interestingly, while HA is necessary, it is not sufficient to turn a strain into a killer, as shown by the results for 1918 HA/NA:Tx/91. It would be interesting to create all possible combinations of 1918:Tx:91 genes and really nail down what is required for the strong pathogenicity of this virus. One final note: The mice were given large doses (1,000,000 viruses) and thus it is no surprise that they all were killed by these viruses. Some may have survived if lower doses were given.
A histopathological analysis was done of the dead mice to determine where the virus had spread to and what damage it had cause. Interestingly, the virus had not spread outside the respiratory tract, and all of its lethal effects were due to damage to the lungs and the creation of a cytokine storm (the release of large amount of immune chemicals in a short period of time) that over excited the immune system of the mice. This in turn caused shock and death.
In the summary, the authors make an important observation..
A number of biological properties associated with this unusually virulent influenza virus were found. Comparison of the 1918 virus with recombinant viruses expressing one or more 1918 virus genes demonstrated that the 1918 HA and polymerase genes are essential for optimal virulence. and that the constellation of all eight genes together make an exceptionally virulent virus in the model systems examined. In fact, no other human influenza viruses that have been tested show a similar pathogenicity for mice 3 to 4 days after infection.
Clearly all 8 genes were necessary for maximum virulence. This might suggest that the lethality shown by the 1918 virus is a very rare event. Only time will tell. The authors also note that this virus is susceptible to antiviral drugs (oseltamivir and amantadine) and vaccines. Indicating that the 1918 virus's lethality does not diminish the effectiveness of health measures we can bring against it, especially vaccines.