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“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 • • • • 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