Download Characterization of the reconstructed 1918 Spanish Influenza

“Characterization of the
reconstructed 1918 Spanish
Influenza Pandemic Virus”
Tumpey et. al.
The Spanish Influenza (1918-1919)
“I had a little bird,
Its name was Enza.
I opened the window,
And in-flu-enza”
Children’s skipping
Rhyme, 1918
Hospital at Camp Funston in Kansas
The Spanish Influenza (1918-1919)
Mortality in
Kansas in October
1918
The Spanish Influenza (1918-1919)
Influenza Virus structure
Hemagglutinin (HA):
glycoprotein at the surface of
the virus. Responsible for
binding the viral cells to the
membrane of human cells.
Neuraminidase (NA):
enzymes antigenic
determinants at the surface
of influenza virus
Polymerase: enzyme that
assemble DNA and RNA
molecules
Specific Question
What made the 1918 Spanish Influenza Virus so
extraordinarily virulent in comparison to the
contemporary human influenza viruses?
What allowed it to replicate in the absence of
trypsin?
Trypsin: Protease, enzyme that cleaves proteins through
hydrolysis of peptide bonds
Methods
 Recovered the 1918 virus from formalin-fixed lungs and frozen
lung tissue from a victim buried in Alaskan permafrost in Nov.
1918
 HA of 1918 virus used in study came from A/S.Carolina/1/18
which has been shown to bind more to human cellular receptors
 Because of its close relation to contemporary H1N1 viruses, they
also took samples of wild-type New Caledonia (N.Cal/99) and
Texas (Tx/91) virus
• Synthesized recombinants of 1918 virus with 1991 Texas H1N1
virus:
 Tx/91 HA:1918 = HA protein from Tx/91, other 7 from 1918
 1918 NA: Tx/91 = NA protein from 1918, other 7 from Tx/91
 1918 HA/NA: Tx/91 = HA and NA from 1918, other 6 from Tx/91
 1918 HA/NA/M/NP/NS: Tx/91 = 5 proteins from 1918, 3 from Tx/91
Study of Virus Infectivity: Methods
 Plaque method: grow layers of cells in serum and insert virus, the
plaques are the voids in the field of cells formed as a result of the
virus killing the cells.
 Studied infectivity of each recombination using Madin-Darby
canine kidney cells with and without presence of trypsin
 Also studied infectivity in human lung epithelial cells inoculated
with each recombinant virus with and without trypsin
 Used Reed and Muench method to find 50% lethal dose (LD50)
which is expressed as logPFU required to give 1 LD50
 PFU = plaque formation unit
 Tested recombinants without the 1918 HA protein and without the
1918 polymerase protein
Study of Virus Infectivity: Results
Plaque formation of the 1918 virus with or without trypsin
Study of Virus Infectivity: Results
Virus titers in human lung tissue cells inoculated with recombinants of the 1918
virus with or without trypsin
Tx/91: black triangle
Tx/91 HA:1918 : white square
1918 HA/NA/M/NP/NS: Tx/91 : black circle
1918 (1): white circle
1918 (2): white triangle
Intranasal inoculation of virus in mice:
Methods
 Intranasally inoculated mice with the different
recombinants of the influenza virus
 Studied the virus titers on day 4 post inoculation
 Calculated morbidity by weight loss
 Also studied the lethality of the virus by finding the 50%
lethal dose for each recombinant
Intranasal inoculation of virus in mice:
Results
Comparison of lung virus titers for different recombinants
Intranasal inoculation of virus in mice:
Results
Comparison of virus lethality
and weight loss for different
recombinants according to days
post inoculation
Tx/91: black triangle
Tx/91 HA:1918 : white square
1918 HA/NA/M/NP/NS: Tx/91 :
black circle
1918 (1): white circle
1918 (2): white triangle
Spread of virus in mice: Methods
 Intranasally inoculated mice with the different
recombinants of the influenza virus
 Studied 50% egg infectious dose in other organs (brain,
heart, liver and spleen) which they harvested on days 4 and
5 post inoculation
 Histopathological analyses of lung tissue from these mice 4
days post inoculation
Spread of virus in mice: Results
50% egg infectious dose in
other organs on days 4 (A)
and 5 (B) post inoculation
Spread of virus in mice: Results
Histopathological images
of lung tissues infected
with the different
recombinants of influenza
virus:
A, B, C: 1918
D: 1918 HA/NA/M/NP/NS:
Tx/91
E: Tx/91 HA:1918
F: Tx/91
Lethality in chicken eggs: Methods
 Inoculated chicken eggs with the different recombinants of
the influenza virus
 Avian H1N1 viruses are extremely lethal to chicken eggs
 Studied lethality recombinants of influenza virus in
comparison to avian H1N1 viruses
Lethality in chicken eggs: Results
Lethality and infectivity of 1918 influenza virus for 10-day old embryonated chicken eggs
MDT = mean time in days for the minimum lethal dose to kill embryos
Implications
 1918 NA is necessary for the virus to replicate without trypsin
because it cleaves HA
 Having both 1918 HA and polymerase allows the virus to be even
more virulent
 It is the specific combination of proteins in the 1918 virus that
makes it so much more virulent and deadly in comparison to
other influenza viruses
 1918 virus specifically attacked lung tissue, did not infect other
organs
 There must also be other proteins in the 1918 virus (other than
HA, NA and polymerase) that make the virus so incredibly
virulent
With this information…
• The 1918 influenza was eradicated, so there was a lot of
controversy bringing it back because of what it could cause.
• Knowing which proteins were involved in making this virus so
pathological is key to understanding the virulence of modern day
influenza viruses and therefore how we can come up against
them
• Further studies could look into more recombinants of 1918 virus
with Tx/91 H1N1 virus to find the additional proteins (other than
HA, NA, and polymerase) that made the Spanish Influenza all the
more virulent
Bibliography
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•

http://virus.stanford.edu/uda/
http://www.rense.com/general63/ebola.htm
http://en.wikipedia.org/wiki/1918_flu_pandemic
http://en.wikipedia.org/wiki/Influenza_A_virus
http://www.sciencemag.org.revproxy.brown.edu/content/suppl/
2005/10/04/310.5745.77.DC1/Tumpey.SOM.pdf
 http://www.mc3cb.com/viruses.html
 http://science.nationalgeographic.com/science/photos/influenza
/
 “Characteristics of the Reconstructed 1918 Spanish Influenza
Pandemic Virus” by Tumpey et. al. (Vol. 310 Science Magazine)
and supplemental materials