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Objectives
- Detection and characterisation of SARS-like CoV
in animals, in China and South-East Asia and in other continents
in zoological related animals (Africa and southern Europe) using
phylogeographic data provided by WP 2 and virological samples gathered
through WP1.
- Description of the global genome evolution of
the SARS CoV, from the earliest patients possible (data provided
through WP8) to latter ones in the course of the epidemic, description
of genetic markers for geographical origins.
- Description of human and animal coronaviruses
belonging to other genetic groups of coronaviruses (OC43 and 229E),
in particular: variable regions and conserved regions with SARS-like
sequences.
Description of work
Task 1:
A/ Design and implementation of an SQL database accessible through
the internet to post data. Field definitions and table structure
with relational links between each other will be conceived to accommodate
all generated sequences as well as all possible strain related data.
B/ Collection and sharing, within WP3 and with WP8, of sequences
data from human SARS CoV from genogroups "CGCCC" (not
M linked) and "TTTTT" (M linked) and all available sequences
of the genome of animal SARS-like viruses already determined.
Task 2:
A/ Design and testing of sets of pairs of primers for the sensitive
detection of SARS-like CoV, including degenerated primers allowing
a less specific detection of viral sequences, based on sequences
published prior to the start of the project.
B/ Design and tests of sets of 30 pairs of primers covering the
whole virus genomes for human, animal SARS-like and non SARS-like
CoV.
Task 3: :
Evaluation of cloning systems after RT-PCR of partial or whole length
genome for genome sequence conservation of viruses not yet passaged
on cell cultures.
Task 4:
A/ Detection of SARS-like CoV in animals of various relevant species,
particularly in masked palm civets sampled in Far East Asia (China
and Vietnam) and Africa or related species (China, Vietnam, Africa
and Southern Europe) by RTPCR using the sets of primers tested in
task 2.
B/ Isolation of CoV from RT-PCR positive samples on cell culture.
Task 5:
Development of serological tests (ELISA, Direct Immunofluorescence,
Western Blot) in homologous and heterologous systems (cross reactivity)
for confirmation of SARS-like CoV infection, particularly for animal
specimens.
Task 6:
Sequencing RT-PCR products or cloned sequences according to the
outcome of tasks 2 and 3.
A/ Sequencing of human viruses from both main genogroups, from Chinese
(Not M linked, human strains with or without deletion in S) and
Vietnamese/French (M linked samples).
B/ Sequencing of animal viruses
C/ Sequencing of human and animal non SARS-like CoV
Task 7:
Sequence assembly and phylogenetic analysis.
Deliverables
D12 Creation of a central secured
SQL database accessible with privileges through the internet to
post all generated sequences
D13 Identification of the animal
species naturally harbouring the SARS-like CoV, which crossed to
humans in 2002, at the root of the ontogenesis of the SARS epidemic
in humans (in connection with WP8).
D14 Identification of SARS-like
viruses in this animal species elsewhere in the world, particularly
in Africa, as well as in zoologically related animal species.
D15 Determination of genetic markers
for animal origins of SARS-like CoV.
D16 Determination of new genetic
markers for geographical origins.
D17 Identification of conserved
and variable regions within the coding part of the genome of the
SARS CoV.
D18 Identification of genomic
regions differing between non SARS CoV and human and animal SARS-like
CoV
D19 Peer-review scientific publications
in international journals
Milestones and expected result
M0-M12:
Collection and sharing of sequences data from human SARS CoV from
genogroups "CGCCC" and "TTTTT" and all available
sequences of the genome of animal SARS-like viruses already determined.
M3-M12:
Development of methods for the partial or complete genome cloning
for virus strains impossible to grow in cell culture.
M12-M24:
Implementation of sequencing strategies
Assessment of the relevance of the detection of SARS-like CoV in
animals of various relevant species, particularly in masked palm
civets sampled in Far East Asia (China and Vietnam) and Africa
M24-M30:
Preparation of manuscripts and report
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