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Cheng Cao Beijing Institute of Biotechnology, China. SAS-Coronavirus: Diagnosis, Antibody Responses and Biosafty Conserns. 2003 SARS Hit the World. Outbroke in Guangdong Province in Nov, 2002 Hundreds Infected with >10% mortality including healthcare workers in 2 months

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SAS-Coronavirus: Diagnosis, Antibody Responses and Biosafty Conserns

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Cheng cao beijing institute of biotechnology china

Cheng CaoBeijing Institute of Biotechnology, China

SAS-Coronavirus: Diagnosis, Antibody Responses and BiosaftyConserns


2003 sars hit the world

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


Sas coronavirus diagnosis antibody responses and biosafty conserns

>8000 Cases

>700 Death


Outbreak contained quickly by intensive public health measure identify patients and isolation

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

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

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


Approaches to develop highly sensitive and specific elisa kit

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.


Sas coronavirus diagnosis antibody responses and biosafty conserns

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

Antigen capturing Vs. Other antibody assays

Sera samples from early phase patients


Antibodies against nucleocapsid protein persisted for years

Antibodies against nucleocapsid protein persisted for years


Sas coronavirus diagnosis antibody responses and biosafty conserns

Thenucleocapsid was involved in

Viral mediated inflammation

Cao et al. 2005

Yasui, et al. 2008


Sas coronavirus diagnosis antibody responses and biosafty conserns

The Nucleocapsid protein inhibits translation by interacting with EF1a


Sas coronavirus diagnosis antibody responses and biosafty conserns

The N protein inhibits cytokinesis

and cell proliferation


Sas coronavirus diagnosis antibody responses and biosafty conserns

The N Protein interacts with Cyp4F3, the

ω-hydroxylase of leukotriene B4


Sas coronavirus diagnosis antibody responses and biosafty conserns

The N Protein inhibits the ω-hydroxylation

of leukotriene B4


Sas coronavirus diagnosis antibody responses and biosafty conserns

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


Sas coronavirus diagnosis antibody responses and biosafty conserns

The N protein activates MASP2, a key

protease in MBL complement pathway


Sas coronavirus diagnosis antibody responses and biosafty conserns

SARS-CoV N protein accelerate the C4 cleavage by MASP2


Sas coronavirus diagnosis antibody responses and biosafty conserns

SARS-CoV N protein play important role in complement activation

C4

C5-9


Sars biosceurity concerns

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 concerns1

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.


Sas coronavirus diagnosis antibody responses and biosafty conserns

谢谢!

Thank you!


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