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Objectives
To study the molecular evolution of SARS CoV within
its human host.
To identify animal and/or human SARS CoV mutations
associated with virulence.
To compare animal and human SARS CoV with respect
to their capacity to bind to, enter and replicate in (primary) target
cells and trigger inflammatory responses.
Description of work
Task 1: SARS CoV viral
sequences obtained from animals (collaboration with WP3), humans
in the early (before the Hotel M outbreak in Hong Kong in March
2003) and late (from March to June 2003) stages of the Chinese epidemic
will be analysed. The sequence information will be compared to epidemiological
data to study the base substitution rate and selection pressure
under different environmental conditions. Genetic signatures characterizing
either the transmission path and/or the evolution of the virus along
the epidemic will be worked out. This information will facilitate
the understanding of the molecular adaptation of the virus to its
human host.
Task 2: In order to identify
mutations associated with virulence or adaptation, molecular variations
(characteristic SNV signatures) among cases of several generations
along the transmission chain from a single-source hospital outbreak
will be studied: PCR+ positive throat clinical samples from patients
belonging to the same outbreak (the General Hospital of Beijing
Armed Police Hospital), categorizing them according to transmission
generation (index case, second generation, third generation, etc.),
age group, co-morbidity, and disease severity (mild, severe) will
be genotyped employing newly developed sensitive methods and compared
across relevant strata.
Task 3: Viral strains
identified as adapted or more virulent will be tested with biochemical
and functional assays to compare their tropism and replicative capacity.
Biochemical assays, e.g. soluble surface proteins, will be used
to study the capacity to bind to primary target cells. Cellular
functional virus entry assays based on HIV pseudotypes bearing SARS
CoV surface proteins S, M and E, will be used to study the capacity
to enter and replicate in human cells. A complimentary assay with
the same objective will be developed by DENG Hongqui at Beijing
University using VSV for pseudotype production. The capacity of
various SARS CoV and individual viral proteins to induce cyto- and
chemokines that could explain the inflammatory response observed
in severe cases of SARS CoV infected patients will be studied using
macro- and microarray technology.
Deliverables
D40 Viral mutations associated
with host adaptation and virulence.
D41 Purified individual surface
proteins S, M and E from animal and human SARS CoV.
D42 Cellular assay for SARS CoV
entry and replication in (primary) human cells.
D43 Cyto- and chemokine induction
profile in cells infected by SARS CoVs or triggered with viral surface
proteins
Milestones and expected result
M12: Identification of mutations
associated with viral adaptation or increased virulence.
M18: Production and purification
of SARS-CoV surface proteins as probes for receptor binding.
M24: Comparative analysis of animal
and human SARS CoV to replicate in (primary) target cells.
M24: Synthesis of SARS CoV pseudotypes
allowing high throuput analysis of the viral entry step.
M30: Identification of differential
receptor usage by animal and human SARS CoV in (primary) human cells.
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