SAS-Coronavirus: Diagnosis, Antibody Responses and Biosafty Conserns Cheng Cao Beijing Institute of Biotechnology, China

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

>8000 Cases >700 Death

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

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

Recombinant Nucleocapsid protein is a good antigen in antibody assay Antigen for coating Antigen For HRP labeling 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).

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.

Patient Type (days after illness) No. of samples No. of Positive % Probable SARS(0-5) Probable SARS(6-10) Probable SARS(10- 61) No SARS fever4400 HCW8000 Healthy people Serum in It seems that the nucleocapsid protein was not cross-reactive with antibodies against other coronavirus Subclinical SARS before outbroke?

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

Antibodies against nucleocapsid protein persisted for years

Cao et al Yasui, et al The nucleocapsid was involved in Viral mediated inflammation

The Nucleocapsid protein inhibits translation by interacting with EF1a

The N protein inhibits cytokinesis and cell proliferation

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

The N Protein inhibits the ω-hydroxylation of leukotriene B4

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

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

SARS-CoV N protein accelerate the C4 cleavage by MASP2

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

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

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.

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