SAS-Coronavirus: Diagnosis, Antibody Responses and Biosafty Conserns

1. Cheng CaoBeijing Institute of Biotechnology, China SAS-Coronavirus: Diagnosis, Antibody Responses and BiosaftyConserns

2. 2003 SARS Hit the World • Outbroke in Guangdong Province in Nov, 2002 • Hundreds Infected with >10% mortality including healthcare workers in 2 months • Great terror spread all-over china • Vinegar sold out in southern china (Anti-viral effect?) • Spread to Beijing and Hong Kong • Etiological agent not confirmed until early April • It is the first time for China to fight against a communicable disease caused by unidentified pathogen

3. >8000 Cases >700 Death

4. Outbreak contained quickly by intensive public health measure (Identify patients and isolation) • Accurate, fast methods for laboratory diagnosis are required • Immunofluorescence Assay (IFA) • RT-PCR • Antibody Assay by ELISA using whole viral lysate as antigen • Antibody Assay by ELISA using recombinant protein as antigen • Antigen (N protein) assay by ELISA using Monoclonal antibody • Only a few BSL-3 labs in China, no accreditation system

5. Immunofluorenscene Assay Antibodies could be detected in 90% patients 15 days after infection No false positive found in 104 health sample (Indirect ELISA using viral lysate gave 2.9% false positive

6. Recombinant Nucleocapsid protein is a good antigen in antibody assay All SARS-CoV encoded proteins are expressed in E.coli Antibodies against N protein could be detected in >80% sera from SARS patients An antigen-capturing ELISA developed in later April, 2003 and certified by SFDA soon No viral culture is needed in the process (Compared to IFA). Antigen for coating Antigen For HRP labeling

7. Approaches to develop highly sensitive and specific ELISA kit • To reduce false positives, highly purified (>99%)protein was employed • Proteins for coating and for labeling were purified by different methods to avoid cross reaction due to bacterial proteins. • The Nucleocapsid protein was expressed as N ter and C ter for coating, to avoid self-aggregating and reduce background.

8. It seems that the nucleocapsid protein was not cross-reactive with antibodies against other coronavirus Subclinical SARS before outbroke?

9. Antigen capturing Vs. Other antibody assays Sera samples from early phase patients

10. Antibodies against nucleocapsid protein persisted for years

11. Thenucleocapsid was involved in Viral mediated inflammation Cao et al. 2005 Yasui, et al. 2008

12. The Nucleocapsid protein inhibits translation by interacting with EF1a

13. The N protein inhibits cytokinesis and cell proliferation

14. The N Protein interacts with Cyp4F3, the ω-hydroxylase of leukotriene B4

15. The N Protein inhibits the ω-hydroxylation of leukotriene B4

16. Expression of N protein resulted in accumulation of LTB4 and lung injury

17. The N protein activates MASP2, a key protease in MBL complement pathway

19. SARS-CoV N protein accelerate the C4 cleavage by MASP2

20. SARS-CoV N protein play important role in complement activation C4 C5-9

22. SARS: Biosceurity Concerns • Reemergence of the disease • SARS-CoV-like virus circulating within the Chinese horseshoe bat • Similar SARS-CoV like virus were identified recently in horseshoe bats in Slovenia and leaf-nosed bats in Nigeria • Laboratory risk • 2004, 8 cases with 1 death due to laboratory leak in Beijing due to inadequate laboratory safety procedure • LAIs have also been reported in laboratory workers in Singapore and Taiwan before

23. SARS: Biosceurity Concerns • Manipulation of SARS coronavirus and infected tissues MUST be done in BSL-3 labs • Only well-trained scientists and technicians could be allowed to work with the virus in BSL-3 lab. Post-graduates in China should not be allowed. • For serum tests, experiments could be done in BSL2 lab. All the sera from SARS patients have to be incubated at 56°C for 30min to inactivate the virus. Sampling should be done in biosafety cabinets.

24. 谢谢！ Thank you！