Transcript Aberrant innate immune response in lethal infection of

Ashley Collins and Amber Cibrario
Kobasa, D., Jones, S. M., Shinya, K., Kash, J. C., Copps, J.,
Ebihara, H., Hatta, Y., Kim, J. H., Halfmann, P., Hatta, M.,
Feldmann, F., Alimonti, J. B., Fernardo, L., Li, Y., Katze, M. G.,
Feldmann, H., and Y. Kawaoka.
• 50 million people died worldwide
• Most deadly to people ages 20 to 40
• Death rate for 15-34-year olds of influenza and pneumonia
were 20 times higher in 1918 than in previous years.
• North America, Europe, Asia, Africa, Brazil, and the South
Pacific
1: Obtain viral RNA from
sample
2: RT-PCR to get cDNA
3: Put cDNA in plasmid
4:Transfect
plasmids into
kidney cell line
5: Cells produce
“new” rescued
virus
Orthomyxoviridae family
- ss(-)RNA virus
- Segmented Genome, carries replicase
- Enveloped
- Major Proteins
- Haemagglutinin (HA)
- Neuraminidase (NA)
- M1 and M2 Matrix Proteins
- 3 types
- Influenza A, B, and C
- Host Range
- Humans, Mammals, Birds
Kawasaki 173 virus
- H1N1 human contemporary influenza virus
- Mild symptomatic virus but causes disease
Cynomolgus Macaque (Crab-eating Macaque)
- Macaca fascicularis
- 93% similarity to human genome
- Relatively easy upkeep in captivity
- Already have been used extensively in
medical research
The purpose of this experiment is to demonstrate a change in the
innate immune system, which may have contributed to the overall
virulence of the 1918 influenza virus.
Objectives
- To demonstrate that 1918 influenza is more virulent
- Virulence maybe result of inflammation
- Demonstrate that innate immune system response is
different compared to control, which may be the
cause of the difference in virulence of the two viruses.
Figure 1A: This figure shows the phage titer at Day 3 in different tissueregions of the macaque for both the 1918 influenza and the control K173.
Figure 1B: This figure shows the phage titer at Day 6 in different
tissue-regions of the macaque for both the 1918 influenza and the
control K173.
Figure 1C: This figure shows the phage titer at Day 8 in different
tissue-regions of the macaque for three of the 1918 influenza and
the control K173.
• Viral titer was much higher in
1918 infected macaques than in
K173 control macaques.
• 1918 virus was able to spread
throughout the lungs and to
other tissues in the body.
Figure 2 Methods
 Tissue Samples
• Fixed in 10% phosphate-buffered formalin.
• 5-µm-thick sections
• Stained with haematoxylin and eosin
• DAKO
Lymphatic
nodule
Alveoli sac
Lung (Panoramic View)
Supplementary Figure 2: Differing lung tissue infected by the control, K173, a
conventional human virus 3 day post-infection.
Figure 2: Differing lung tissue infected by the control, K173, a conventional
human virus 8 day post-infection.
K173- 3 days
post infection
K173- 8 days
post infection
Figure 2: Comparison of infected K173 lung tissue samples at 3 days and 8 days post
infection.
Supplementary Figure 2: Differing lung tissue infected by 1918 influenza
virus 3 day post-infection.
Figure 2: Differing lung tissue infected by the 1918 influenza virus in
macaques.
1918 -3 days
post infection
1918-8 days
post infection
Figure 2: Comparison of 1918 infected lung tissue samples at 3 days and 8 days post
infection.
Figure 2: Comparing K173 infected lung
tissue to 1918 infected lung tissue 3 day
post-infection.
Figure 2: Comparing K173 infected lung tissue to 1918 infected lung tissue 8
day post-infection.
1918 Infected Lungs
K173 Infected Lungs
• 1918 influenza had caused much greater Tissue Damage.
• Tissure Damage may be result of inflammation.
Supplementary Figure 1: Gross Pathology of lungs on day 8 post infection.
a.) 1918 infected lungs and b.)K173 infected lungs.
Cytokines and Chemokines
- CCL5/CCL2: recruitment of
leukocytes and inflammatory
response
- IL-8: phagocyte chemotaxis and
activation
- IL-6: acts as pro-inflammatory and
anti-inflammatory. Regulates
fever and acute phase response
Cytometric Bead Array assay (CBA)
Human Chemokine CBA kit
Human IL-6 Flex set
FCAP software to analyze
Used with permission of Becton,
Dickinson and Company
Figure 3: Levels of chemokines and cytokines in blood serum of infected
macaques of both the control K173 and the 1918 influenza virus over 8 days.
• Cytokine IL-6 levels were elevated in blood
serum.
• May have cytokine storm
•They want to look at the immune system as a
whole may show change in innate immune
response.
• This change may contribute to viral virulence.
• Type of DNA array
•PCR (polymerase chain reaction) amplified gene fragments fixed on a
glass slide (chip)
•Samples are either RNA or cDNA
•Samples to be analyzed are labeled with a fluorescent dye
•Cy3/Cy5
•Samples are added to the chip and hybridize to appropriate spot
•The level of fluorescence is proportional to the level of gene
expression.
•Oligonucleotide microarray
•The chip contained ~22,000 genes representing ~18,000 genes.
•Samples contained equal masses of total RNA isolated from bronchi from
infected macaques
•Samples were amplified using Low RNA Input Linear Amplification Kit
(Agilent Technology) per manufacturers instructions
• Reverse transcription PCR to produce cDNA
•Add fluorescently labeled DNA nucleotides bought from manufacturer at
the time of reverse transcription (Kash, J. C. et. al, 2006).
•Global gene expression from the bronchi was compared to RNA prepared
in the same way from lung tissue of three mock infected macaques.
•Raw microarry data was processed by Feture Extraction 8.1 software
(Agilent Technology)
•Submitted to a database (Expression Array Manager)
•Analyzed by Rosetta Resolver System 5.1 (Rosetta Biosoftware) and
Spotfire Decision Site for Functional Genomics 8.1 (Spotfire).
•Observe possible differences in regulation of global and
immune related genes.
•Clues to virulence.
•Shows overall gene expression profiles.
•Shows differenced between individuals infected with the
1918 virus and how the data was averaged.
Figure 4 | Microarray analysis of gene expression in a bronchi of macaques on
days 3, 6, and 8 post-infection. a, Overall gene expression profiles in K173- and
1918-virus-infected animals.
•By observing the expression of chemokines and cytokines in K173- and
1918-infected macaques differences can be observed in the immune
response.
•If the 1918 flu virus effects the immune system differently than the K173
virus then chemokine and cytokine genes will be regulated differently.
Figure 4 | Microarray analysis of gene expression in a bronchi of macaques on
days 3, 6, and 8 post-infection. b, Expression of chemokines and cytokines as
determined by expression oligonucleotide microarray analysis of bronchial tissue
from macaques infected with the K173 or 1918 influenza virus.
•Shows expression of type I interferon stimulated genes.
•Shows any more differences in response to the two
viruses.
Figure 4 | Microarray analysis of gene
expression in a bronchi of macaques on days 3,
6, and 8 post-infection.
•The resulting microarray depicts the array of all the genes responsible
for the immune system response that were represented in the global
array.
•Gene ontology: The grouping of related genes responsible for a related
function
• Also grouped genes responsible for carbohydrate
metabolism, cellular lipid metabolism, and generation of
precursor metabolites and energy.
Figure 1 shows us that the level of viral titer is high from day 3-8 post infection and does not
drop or change much. It also shows us that the 1918 virus is able to spread throughout the
respiratory system and even to other tissues in the body.
Figure 2 shows us that tissue damage in the 1918 infected macaques is much more severe and
spread out throughout the respiratory system. Also the main damage of the tissue seems to
be caused by inflammation and aspects of the innate immune system.
Figure 3 shows us that the level of IL-6 cytokine is very high, which may result in the change
of innate immunity. This also may result in high levels of inflammation over a long period
of time conferring figure 2.
Figure 4 shows us that the virulence of the 1918 flu virus probably results from down
regulation of genes important for recruiting T-cells and macrophages and up regulation of
inflammatory genes. This results in a longer amount of time for the virus to replicate and
take hold of the tissue and produces a large amount of inflammation causing sever tissue
damage. The delay in the innate immune system seems to be just long enough so that when
the immune system does become activated it can’t catch up before the individual dies.
Fodor, E., et al. 1999. Rescue of Influenza A Virus from Recombinant DNA. J. of Viro. 73:9679-9682.
Hartwell, L., L. Hood, M. Goldberg, A. Reynolds, L. M. Silver, R. C. Veres. 2001. Genetics from genes to genomes. 340342. McGraw-Hill, New York.
Kash, J. C. et al. 2006 Global suppression of the host antiviral response by Ebola- and associated with enhanced virulence.
J. Virol. 80:3009-3020.
Kobasa, D., Jones, S. M., Shinya, K., Kash, J. C., Copps, J., Ebihara, H., Hatta, Y., Kim, J. H., Halfmann, P., Hatta, M.,
Feldmann, F., Alimonti, J. B., Fernardo, L., Li, Y., Katze, M. G., Feldmann, H., and Y. Kawaoka. 2007.
Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus. Nature
445: 319-323.
Pekosz, A., et al. 1999. Reverse genetics on negative-strand RNA viruses: Closing the circle. Proc. Natl. Acad. Sci. USA.
96:8804-8806.
Human Genome Sequencing Center (13 April 2007). "DNA sequence of Rhesus macaque has
evolutionary, medical implications". Press release. Retrieved on 2008-04-28.