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

Antigenfor coating

AntigenFor 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) 24 4 14.8

Probable SARS(6-10) 38 26 68.4

Probable SARS(10-61)

135 121 89.6

No SARS fever 44 0 0

HCW 80 0 0

Healthy people 940 1 0.106

Serum in 2002 1800 1 0.06

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. 2005Cao et al. 2005 Yasui, et al. 2008Yasui, et al. 2008

The nucleocapsid was involved inViral 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-hydroxylationof leukotriene B4

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

The N protein activates MASP2, a keyprotease in MBL complement pathway

SARS-CoV N protein accelerate the C4 cleavage by MASP2

SARS-CoV N protein play important role in complement activation

C4C4 C5-9C5-9

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.

谢谢！