Transcript SARS (and a touch of Proteomics)

SARS (and a touch of
Proteomics)
Greg Zornetzer
Project Summary
10/14/2008
SARS Outbreak Started in Guangdong in 2002 and
Progressed to 26 Countries
Epidemic Curve
Phylogenetic tree of triphasic SARS epidemic
Late Phase
Middle Phase
Civet cat
Early Phase
Horseshoe Bat
Chinese SARS Molecular Epidemiology Consortium, Science, 2004
Poutanen and Low, Curr. Opin. Infect. Dis., 2004
“Features” of SARS-CoV
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Coronavirus
Enveloped
ss(+)RNA genome
Receptor is ACE2
SARS-related
downregulation of
ACE2 contributes to
lung damage
Macaque Model System
• By our collaborators – Osterhaus group
• Pathological changes in the lung,
especially in aged animals
• Challenges:
– Virus is not lethal
– High degree of animal/animal variation
Ongoing Experiments
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Timecourse experiment in aged macaques
Serial blood draws (days 1, 2, and 4)
Sacrifice animals (days 1, 2, and 4)
3 animal replicates, with aged mocks
• Question: Do PBMC studies (possible in
human disease) inform on the state in the
lungs?
Macaque SARS experiments
• Can we make a more pathogenic virus?
• Passage in vivo in macaques (Rotterdam)
– Infect animals
– Harvest virus at peak replication
– Repeat
• Passage in vitro in macaque bronchial
cells (UNC)
SARS in Mice
• Collaboration with Ralph Baric
• Young mice infected with Urbani support
replication, but no significant disease
• Aged mice infected with Urbani exhibit
weight loss & lung pathology, but
ultimately survive infection
• Aged mice infected with modified Urbani
viruses exhibit fatal disease
– Kinetics of the immune response are critical
MA15: Kills young Balb/C mice
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ORF1a
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ORF1b
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• 3 viruses (MA15, 2 attenuated MA15
“hybrids”)
• Balb/C mice – 4 animal replicates
• Time points: 12 hrs, 1 day, 2 days, 4 days
• What makes MA15 a “killer” virus in mice?
MA15 knockout studies
• MA15 causes disease but not death in
C57/BL6 mice and 129 mice
• Ifn a/b Receptor KO or Ifn g Receptor KO
mice do not exhibit enhanced disease
• STAT-1 KO mice die after 9 days of
fibrosis and viral replication
Collaborative Cross Mice
• Project to develop many inbred lines that
embody most of mouse genetic diversity
• We can dig down and examine the genetic
basis for enhanced or reduced disease
• This approach uses a LOT of mice & can
generate a LOT of data.
SARS in Primary Cells
• Infection of Human Airway Epithelial cells
• Infection of Macaque Bronchial cells
• Possible stimulation of immune cells with
SARS-CoV
Proteomics
• Personal interest in protein-protein
interactions
• Use pulldowns + Mass Spectrometry
• But we must keep our minds open to other
approaches: Y2H, crosslinking, nonpulldown methods
Host Responses Activated by
dsRNA and RNA Viruses
Adapted from Gale, M Jr. and Foy, EM. (2005) Nature. 436:939-45.
Finding IPS-1 Interacting Proteins
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Mitochondrial proteomics
+/- IPS-1
+/- Virus stimulation
Survey the mitochondrion for protein
population changes