1. SARS (and a touch of Proteomics) Greg Zornetzer Project Summary 10/14/2008

2. SARS Outbreak Started in Guangdong in 2002 and Progressed to 26 Countries SARS Outbreak Started in Guangdong in 2002 and Progressed to 26 Countries Chinese SARS Molecular Epidemiology Consortium, Science, 2004 Poutanen and Low, Curr. Opin. Infect. Dis., 2004 Epidemic Curve Civet cat Phylogenetic tree of triphasic SARS epidemic Early PhaseMiddle Phase Late Phase Horseshoe Bat

3. “Features” of SARS-CoV Coronavirus Enveloped ss(+)RNA genome Receptor is ACE2 SARS-related downregulation of ACE2 contributes to lung damage

4. 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

5. Ongoing Experiments 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?

6. 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)

7. 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

8. MA15: Kills young Balb/C mice 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? S ORF1a ORF1b E M N 1 2 3 4 5 ** * * * *

9. MA15 knockout studies MA15 causes disease but not death in C57/BL6 mice and 129 mice Ifn  Receptor KO or Ifn  Receptor KO mice do not exhibit enhanced disease STAT-1 KO mice die after 9 days of fibrosis and viral replication

10. 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.

11. SARS in Primary Cells Infection of Human Airway Epithelial cells Infection of Macaque Bronchial cells Possible stimulation of immune cells with SARS-CoV

12. Proteomics Personal interest in protein-protein interactions Use pulldowns + Mass Spectrometry But we must keep our minds open to other approaches: Y2H, crosslinking, non- pulldown methods

13. Host Responses Activated by dsRNA and RNA Viruses Adapted from Gale, M Jr. and Foy, EM. (2005) Nature. 436:939-45.

14. Finding IPS-1 Interacting Proteins Mitochondrial proteomics +/- IPS-1 +/- Virus stimulation Survey the mitochondrion for protein population changes