sas-coronavirus: diagnosis, antibody responses and biosafty conserns cheng cao beijing institute of...
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SAS-Coronavirus: Diagnosis, Antibody
Responses and Biosafty Conserns
Cheng Cao
Beijing Institute of Biotechnology, China
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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
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>8000 Cases
>700 Death
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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
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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
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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).
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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.
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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?
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Antigen capturing Vs. Other antibody assays
Sera samples from early phase patients
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Antibodies against nucleocapsid protein persisted for years
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Cao et al. 2005Cao et al. 2005 Yasui, et al. 2008Yasui, et al. 2008
The nucleocapsid was involved inViral mediated inflammation
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The Nucleocapsid protein inhibits translation by interacting with EF1a
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The N protein inhibits cytokinesis and cell proliferation
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The N Protein interacts with Cyp4F3, the ω-hydroxylase of leukotriene B4
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The N Protein inhibits the ω-hydroxylationof leukotriene B4
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Expression of N protein resulted in accumulation of LTB4 and lung injury
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The N protein activates MASP2, a keyprotease in MBL complement pathway
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SARS-CoV N protein accelerate the C4 cleavage by MASP2
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SARS-CoV N protein play important role in complement activation
C4C4 C5-9C5-9
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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
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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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谢谢!