FR2862981A1 - New isolated and purified strain of coronavirus associated with severe acute respiratory syndrome, useful for preparing diagnostic reagents and vaccines, also derived proteins, nucleic acids and antibodies - Google Patents

Abstract

An isolated and purified strain of human coronavirus (CV) associated with severe acute respiratory syndrome (SARS), is new. Isolated and purified strain of human coronavirus (CV) associated with severe acute respiratory syndrome (SARS). Its genome, in the form of cDNA, includes a Ser codon at positions 23220-23222 of the S protein or a Gly codon at positions 25298-25300 of the ORF3 gene, and an Ala codon at positions 7918-7920 of ORF1a or a Ser codon at positions 26857-26859 of the protein M gene, where all positions are referenced to the Genbank sequence AY27419.3. Independent claims are also included for: (1) isolated or purified polynucleotide (I) from the genome of the new strain; (2) isolated or purified polynucleotide (Ia) that hybridizes to (I) under highly stringent conditions; (3) fragments of (I) or (Ia); (4) pairs of primers that can amplify part of the genome of CV, or its equivalent DNA; (5) probes that can detect the genome of CV, or part of it; (6) DNA or RNA chips or filters that contain at least one of (I), (Ia) or their fragments; (7) recombinant cloning and/or expression vector that contains a fragment of (3); (8) cDNA bank that comprises the fragments of (3); (9) cells modified by the vector of (7) or the bank of (8); (10) isolated, purified protein or peptide (II) that is encoded by (I), (Ia) or their fragments; (11) mono- or poly-clonal antibodies (Ab), or their fragments, prepared by immunization with the vector of (7), the bank of (8) or (II), and able to bind to at least one (II); (12) protein or peptide chip or filter that includes at least one (II), Ab or fragment of Ab; (13) detection reagent for a SARS-associated CV that contains the new primers, probes, chips, filters, vectors, CV strain, (I), (Ia), (II), Ab or its fragments; (14) detecting CV by amplifying, from material extracted from a sample, a fragment of ORF-N by reverse transcription PCR, using the primers of (4), then detecting any amplicon formed; and (15) kit for detection of CV comprising any of the new primers, probes, DNA/RNA chips or filters, vectors, cells, isolated CV, (I) or (Ia). ACTIVITY : Virucide. No biological data given. MECHANISM OF ACTION : Vaccine.

Description

2862981 1

  The present invention relates to a new strain of coronavirus associated with severe acute respiratory syndrome (SARS), resulting from a sample recorded under No. 031589 and taken in Hanoi (Vietnam), from nucleic acid molecules derived from its genome , the proteins and peptides encoded by said nucleic acid molecules and their applications, especially as diagnostic reagents and / or as a vaccine.

  The coronavirus is a single-stranded, positive polarity, approximately 30 kilobase RNA virus that replicates in the cytoplasm of host cells; the 5 'end of the genome has a cap structure and the 3' end has a polyA tail. This virus is enveloped and includes, on its surface, -peplomeric structures-called spicules.

  The genome comprises the following open reading frames or ORFs, from its 5 'end to its 3' end: ORF1a and ORF lb corresponding to the proteins of the transcription-replication complex, and ORF-S, ORF-E, ORFM and ORF- N corresponding to structural proteins S, E, M and N. It also includes ORFs corresponding to proteins of unknown function encoded by: the region between ORF-S and ORF-E and overlapping it, the region located between ORF-M and ORF-N, and the region included in the ORF-N.

  Protein S is a membrane glycoprotein (200-220 kDa) that is in the form of spikes or "spike" emerging from the surface of the viral envelope. It is responsible for the attachment of the virus to the receptors of the host cell and the induction of viral envelope fusion with the cell membrane.

  The small envelope protein (E) also called sM (small membrane), which is an unglycosylated trans-membrane protein of about 10 kDa, is the protein present in a smaller amount in the virion. It plays a driving role in the process of budding of coronaviruses that occurs at the level of the intermediate compartment in the endoplasmic reticulum and the Golgi apparatus. The protein M or matrix protein (25-30 kDa) is a membrane glyco-protein more abundant that is integrated into the viral particle by an MIE interaction, while the incorporation of S into the particles is directed by an S / M interaction. It appears to be important for the viral maturation of coronavirus 2862981 2 and for the determination of the site at which the virus particles are assembled.

  N protein or nucleocapsid protein (45-50 kDa), which is the most conserved among the structural proteins of coronaviruses, is necessary to encapsidate the genomic RNA and then to direct its incorporation into the virion. This protein is also likely to be involved in RNA replication.

  When a host cell is infected, the reading frame (ORF) located 5 'of the viral genome is translated into a polyprotein which is cleaved by the viral proteases and then releases several nonstructural proteins such as RNA polymerase RNA dependent (Rep) and ATPase helicase (Hel). Both proteins are involved in the replication of the viral genome as well as in the generation of transcripts that are used in the synthesis of viral proteins. The mechanisms by which these sub-genomic mRNAs are produced are not fully understood; however, recent evidence indicates that the transcriptional regulatory sequences at the 5 'end of each gene represent signals that regulate the discontinuous transcription of sub-genomic mRNAs.

  The proteins of the viral membrane (S, E and M proteins) are inserted into the intermediate compartment, whereas the replicated RNA (+ strand) assembles with the N protein (nucleocapsid). This protein-RNA complex then associates with the M protein included in the endoplasmic reticulum membranes and the viral particles are formed when the core nucleocapsid complex buds into the endoplasmic reticulum. The virus then migrates through the Golgi complex and eventually exits the cell, for example by exocytosis. The site of attachment of the virus to the host cell is at the level of the S protein. Coronaviruses are responsible for 15 to 30% of colds in humans and respiratory or digestive infections in animals, including cats. (FIPV: Feline infectious peritonitis virus), poultry (IBV: Avian Infectious bronchitis virus), mouse (MHV: Mouse Hepatitis virus), pig (TGEV: Transmissible gastroenteritis virus, PEDV: Porcine Epidemic Diarrhea virus, PRCoV: Porcine Respiratory Coronavirus, HEV: Hemagglutinating encephalomyelitis Virus) and cattle (BcoV: Bovine coronavirus).

  In general, each coronavirus affects only one species; in immunocompetent individuals, the infection induces possibly neutralizing antibodies and cellular immunity, capable of destroying the infected cells.

  An epidemic of atypical pneumonia, known as SARS (Severe Acute Respiratory Syndrome, SARS in French) has spread to different countries (Vietnam, Hong Kong, Singapore, Thailand and Canada) during the first trimester. 2003, from an initial outbreak appeared in China in the last quarter of 2002. The severity of this disease is such that its mortality rate is about 3 to 6%. The determination of the causative agent of this disease has been undertaken by many laboratories around the world.

  In March 2003, a new coronavirus (SARS-CoV, SARS virus or SARS virus, in French) was isolated, in association with cases of severe acute respiratory syndrome (TGKSIAZEK et al., The New England Journal of Medicine, 2003 , 348, 1319-1330, C. DROSTEN et al., The New England Journal of Medicine, 2003, 348, 1967-1976, Peiris et al., Lancet, 2003, 361, 1319-).

  Genome sequences of this new coronavirus have thus been obtained, in particular those of the Urbani isolate (Genbank n access AY274119.3 and A. MARRA et al., Science, May 1, 2003, 300, 1399-1404) and of the Toronto isolate (Tor2, Genbank Access No. AY 278741 and A. ROTA et al., Science, 2003, 300, 1394-1399).

  The organization of the genome is comparable to that of other known coronaviruses, thus confirming the SARS-CoV membership of the family Coronaviridae; the open reading frames ORFla and lb and the open reading frames corresponding to the S, E, M, and N proteins, as well as proteins encoded by: the region between the ORF-S and the ORF-E (ORF3), the region between the ORF-S and the ORF-E and straddling the ORF-E (ORF4), the region between the ORF-M and the ORF-N (ORF7 to ORF 11 ) and the region corresponding to ORF-N (ORF 13 and ORF 14) have been identified.

  Seven differences were found between the sequences of isolates Tor2 and Urbani; 3 correspond to silent mutations (c / t in position 16622 and a / g in position 19064 of ORFlb, t / c in position 24872 of ORF-S) and 4 modify the amino acid sequence of respectively: proteins encoded by the ORFla (c / t at position 7919 corresponding to the A / V mutation), the S protein (g / t at position 23220 corresponding to the A / S mutation), the protein encoded by ORF3 (a / g at position 25298 corresponding to the R / G mutation) and the M protein (t / c at position 26857 corresponding to the SIP mutation).

  In addition, phylogenetic analysis shows that SARS-CoV is far from other coronaviruses and has been shown to be either by mutation of human respiratory coronaviruses or by recombination between known coronaviruses (for a review, see Holmes, JCI, 2003, 111, 1605-1609).

  The identification and consideration of novel variants is important for the development of sufficiently sensitive and specific SARS detection and diagnostic reagents as well as for immunogenic compositions capable of protecting populations against SARS outbreaks.

  The inventors have now identified another strain of SARS-associated coronavirus, which is different from the Tor2 and Urbani isolates.

  The subject of the present invention is therefore an isolated or purified strain of human coronavirus associated with severe acute respiratory syndrome, characterized in that its genome presents in the form of complementary DNA a serine codon at position 23220-23222 of the protein gene. S or a glycine codon at position 25298-25300 of the ORF3 gene, and an alanine codon at position 7918-7920 of ORFIa or a serine codon at position 26857-26859 of the M protein gene, said positions being indicated with reference to Genbank sequence AY274119.3.

  According to an advantageous embodiment of said strain, the DNA equivalent of its genome has a sequence corresponding to the sequence SEQ ID NO: 1; this strain of coronavirus is derived from the bronchoalveolar lavage sample of a patient with SARS, listed under the number 031589 and carried out at the French hospital in Hanoi (Vietnam).

  According to the invention, said sequence SEQ ID NO: 1 is that of the deoxyribonucleic acid corresponding to the ribonucleic acid molecule of the genome of the strain isolated from coronavirus as defined above.

  The sequence SEQ ID NO: 1 differs from the Genbank sequence AY274119.3 (isolate Tor2) in that it possesses the following mutations: 2862981 5 g / t at position 23220; the alanine codon (gct) at position 577 of the amino acid sequence of the Tort protein S is replaced by a serine codon (tct), a / g at position 25298; the arginine (aga) codon at position 11 of the amino acid sequence of the Tor 2 ORF3 encoded protein is replaced by a glycine codon (gga).

  In addition, the sequence SEQ ID NO: 1 differs from the Genbank sequence AY278741 (Urbani isolate) in that it possesses the following mutations: tic at position 7919; the valine codon (gtt) at position 2552 of the amino acid sequence of the protein encoded by ORF 1a is replaced by an alanine codon (gct), - t / c at position 16622: this mutation does not modify the sequence in amino acids of the proteins encoded by the ORFIb (silent mutation), - g / a in position 19064: this mutation does not modify the amino acid sequence of the proteins encoded by the ORFIb (silent mutation), - c / t in position 24872: this mutation does not modify the amino acid sequence of protein S, and - c / t at position 26857: the proline codon (ccc) at position 154 of the amino acid sequence of protein M is replaced by a serine codon (tcc).

  In the absence of particular mention, the positions of the nucleotide and peptide sequences are indicated with reference to the Genbank sequence AY274119.3.

  The present invention also relates to an isolated or purified polynucleotide, characterized in that its sequence is that of the genome of the isolated strain of coronavirus as defined above.

  According to an advantageous embodiment of said polynucleotide, it has the sequence SEQ ID NO: 1.

  The present invention also relates to an isolated or purified polynucleotide, characterized in that its hybrid sequence under conditions of high stringency with the polynucleotide sequence as defined above.

  The terms isolated or purified mean modified by the hand of man from the natural state; that is, if an object exists in nature, it is said to be isolated or purified if it has been modified or extracted from its natural environment or both. For example, a polynucleotide or protein / peptide naturally present in a living organism is neither isolated nor purified; on the other hand, the same polynucleotide or protein / peptide separated from the coexisting molecules in its natural environment, obtained by cloning, amplification and / or chemical synthesis, is isolated within the meaning of the present invention. In addition, a polynucleotide or a protein / peptide that is introduced into an organism by transformation, genetic manipulation or any other method is isolated even if it is present in said organism. The purified term as used in the present invention means that the proteins / peptides according to the invention are essentially free from association with other proteins or polypeptides, as is for example the purified product of the host cell culture. recombinant or the purified product from a non-recombinant source.

  Within the meaning of the present invention, high-stringency hybridization conditions are understood to mean temperature and ionic strength conditions chosen in such a way as to allow the maintenance of specific and selective hybridization between complementary polynucleotides.

  By way of illustration, conditions of high stringency for the purpose of defining the above polynucleotides are advantageously as follows: DNA-DNA or DNA-RNA hybridization is carried out in two stages: (1) prehybridization at 42 C during 3 hours in phosphate buffer (20 mM pH 7.5) containing 5 x SSC (1 x SSC corresponds to a solution 0.15 M NaCl + 0.015 M sodium citrate), 50% formamide, 7% sodium dodecyl sulfate (SDS), Denhardt's x 10, 5% dextran sulfate and 1% salmon sperm DNA; (2) hybridization for 20 hours at 42 ° C. followed by 2 washes for 20 minutes at 20 ° C. in 2 × SSC + 2% SDS, 1 washing for 20 minutes at 20 ° C. in 0.1 × SSC + 0.1% SDS. The last washing is carried out in 0.1 x SSC + 0.1% SDS for 30 minutes at 60 C.

  The subject of the present invention is also a representative fragment of the polynucleotide as defined above, characterized in that it is capable of being obtained, either by the use of restriction enzymes whose recognition sites and cleavage are present in said polynucleotide as defined above, either by amplification using oligonucleotide primers specific for said polynucleotide as defined above, either by in vitro transcription or by chemical synthesis.

  According to an advantageous embodiment of said fragment, it is selected from the group consisting of: the cDNA corresponding to at least one open reading frame (ORF) chosen from: ORF 1a, ORF 1b, ORF-S, ORF-1 E, ORF-M, ORF-N, ORF3, ORF4, ORF7 to ORF11, ORF13 and ORF14, and the cDNA corresponding to the 5 'or 3' non-coding ends of said polynucleotide.

  According to an advantageous arrangement of this embodiment, said fragment has a sequence selected from the group consisting of: the sequences SEQ ID NO: 2 and 4 representing the cDNA corresponding to the ORF-S which encodes the protein S, - the sequences SEQ ID NO: 13 and 15 representing the cDNA corresponding to the ORF-E which codes for the protein E, the sequences SEQ ID NO: 16 and 18 representing the cDNA corresponding to the ORF M coding for the M protein, the sequences SEQ ID NO: 36 and 38 representing the cDNA corresponding to the ORF-N which encodes the N protein, the sequences representing the corresponding cDNAs respectively to the ORFIa and ORFIb (ORF1ab, SEQ ID NO: 31), ORF3 and ORF4 (SEQ ID NO: 7, 8), ORFs 7-11 (SEQ ID NO: 19, 20), ORF13 (SEQ ID NO: 32) and ORFI4 (SEQ ID NO: 34), and the sequences representing the cDNAs corresponding to the 5 '(SEQ ID NO: 39 and 72) and 3' non-coding (SEQ ID NO: 40, 73) of said polynucleotide.

  The subject of the present invention is also a fragment of the cDNA coding for the protein S, as defined above, characterized in that it has a sequence selected from the group consisting of the sequences SEQ ID NO: 5 and 6 (fragments Sa and Sb).

  The subject of the present invention is also a fragment of the cDNA corresponding to the ORFla and ORF Ib as defined above, characterized in that it has a sequence selected from the group consisting of the sequences SEQ ID NO: 41 to 54 (fragments LO to L12).

  The present invention also relates to a fragment of the polynucleotide as defined above, characterized in that it has at least 15 bases or consecutive base pairs of the genome sequence of said strain including at least one of those located in position 7979, 16622, 19064, 23220, 24872, 25298 and 26857. Preferably, it is a 20 to 2500 base or base pair fragment, preferably 20 to 400.

  According to an advantageous embodiment of said fragment, it includes at least one pair of bases or base pairs corresponding to the following positions: 7919 and 23220, 7919 and 25298, 16622 and 23220, 19064 and 23220, 16622 and 25298, 19064 and 25298 , 23220 and 24872, 23220 and 26857, 24872 and 25298, 25298 and 26857.

  The subject of the present invention is also primers of at least 18 bases capable of amplifying a fragment of the genome of a SARS-associated coronavirus or the DNA equivalent thereof.

  According to one embodiment of said primers, they are selected from the group consisting of: - primer pair n 1 corresponding respectively to positions 28507 to 28522 (sense primer, SEQ ID NO: 60) and 28774 to 28759 (primer antisense, SEQ ID NO: 61) of the polynucleotide sequence as defined above, and - primer pair n 2 corresponding to positions 28375 to 28390 (sense primer, SEQ ID NO: 62) and 28702 respectively. to 28687 (antisense primer, SEQ ID NO: 63) of the polynucleotide sequence as defined above.

  The subject of the present invention is also a probe capable of detecting the presence of the genome of a SARS-associated coronavirus or of a fragment thereof, characterized in that it is selected from the group consisting of: fragments such as as defined above and the fragments corresponding to the following positions of the polynucleotide sequence as defined above: 28561 to 28586, 28588 to 28608, 28541 to 28563 and 28565 to 28589 (SEQ ID NO: 64 to 67).

  The probes and primers according to the invention may be labeled directly or indirectly by a radioactive or non-radioactive compound by methods well known to those skilled in the art, in order to obtain a detectable and / or quantifiable signal. Among the radioactive isotopes used, mention may be made of 32P, 33P, 2862981, 355, 3H or 125I. Non-radioactive entities are selected from ligands such as biotin, avidin, streptavidin, digoxygenin, haptens, dyes, luminescent agents such as radioluminescent, chemiluminescent, bioluminescent, fluorescent, phosphorescent agents.

  The invention encompasses probes and labeled primers derived from the foregoing sequences.

  Such probes and primers are useful for the diagnosis of SARS-associated coronavirus infection.

  The present invention also relates to a method for detecting a SARS-associated coronavirus from a biological sample, which method is characterized by comprising at least: nucleic acids present in said biological sample, (b) amplification of a fragment of ORF-N by RT-PCR using a primer pair as defined above, and c) the detection by any appropriate means of the amplification products obtained in (b).

  The amplification products (amplicons) in (b) are 268 bp for primer pair n 1 and 328 bp for primer pair n 2.

  According to an advantageous embodiment of said method, the detection step (b) is carried out using at least one probe corresponding to positions 28561 to 28586, 28588 to 28608, 28541 to 28563 and 28565 to 28589 of the sequence of the polynucleotide as defined above.

  Preferably, the SARS-associated coronavirus genome is detected and possibly quantified by real-time PCR, using primer pair n 2 and probes corresponding to positions 28541 to 28563 and 28565 to 28589 labeled with compounds. different, including different fluorescent agents.

  Real-time RT-PCR which uses this pair of primers and this probe is very sensitive since it makes it possible to detect 102 copies of RNA and up to 10 RNA copies, it is moreover reliable and reproducible.

  The invention encompasses polydeoxyribonucleotides and single-stranded, double-stranded and tripe-stranded polyribonucleotides corresponding to the genome sequence of the isolated strain of coronavirus and its fragments as defined above, as well as to their complementary sequences, sense or antisense, in particular RNAs and cDNAs corresponding to the sequence of the genome and its fragments as defined above.

  The present invention also encompasses the amplification fragments obtained using primers specific to the genome of the purified or isolated strain as defined above, in particular using primers and primer pairs such as defined above, the restriction fragments constituted by or comprising the sequence of the fragments as defined above, the fragments obtained by in vitro transcription from a vector containing the sequence SEQ ID NO: 1 or a fragment such as defined above, as well as fragments obtained by chemical synthesis. Examples of restriction fragments are deduced from the restriction map of the sequence SEQ ID NO: 1 illustrated in FIG. 13. In accordance with the invention, said fragments are either in the form of isolated fragments or in the form of mixtures of fragments. . The invention also encompasses modified fragments, relative to the above, by removal, or addition of nucleotides in a proportion of about 15%, relative to the length of the fragments above and / or modified at the nature of the nucleotides, since the modified nucleotide fragments retain a hybridization capacity with the genomic or antigenome RNA sequences of the isolate as defined above.

  The nucleic acid molecules according to the invention are obtained by conventional methods, known in themselves, following the standard protocols such as those described in Current Protocols in Molecular Biology (Frederick M. A USUBEL, 2000, Wiley and his Inc, Library of Congress, USA). For example, they can be obtained by amplification of a nucleic sequence by PCR or RTPCR or by total or partial chemical synthesis.

  The present invention also relates to a DNA or RNA chip or filter, characterized in that it comprises at least one polynucleotide or one of its fragments as defined above.

  The chips or DNA or RNA filters according to the invention are prepared by conventional methods, known per se, such as, for example, chemical or electrochemical grafting of oligonucleotides on a glass or nylon support.

  The subject of the present invention is also a vector for cloning and / or recombinant expression, in particular a plasmid or a phage comprising a nucleic acid fragment as defined above. Preferably, said recombinant vector is an expression vector wherein said nucleic acid fragment is under the control of appropriate transcriptional and translational regulatory elements. In addition, said vector may comprise sequences (labels or tag) fused in phase with the 5 'and / or 3' end of said insert, useful for the immobilization, and / or the detection and / or purification of the protein expressed from said vector.

  These vectors are constructed and introduced into host cells by conventional methods of recombinant DNA and genetic engineering, which are known per se. Many vectors in which a nucleic acid molecule of interest can be inserted in order to introduce and maintain it in a host cell are known per se; the choice of an appropriate vector depends on the use envisaged for this vector (for example replication of the sequence of interest, expression of this sequence, maintenance of the sequence in extrachromosomal form or integration into the chromosomal material of the host ), as well as the nature of the host cell.

  According to the invention, said plasmid is in particular selected from the following plasmids: the plasmid, called SARS-S, included in the bacterial strain deposited under the No. I-3059, on June 20, 2003, from the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15; it contains the cDNA sequence coding for the S protein of the strain of SARS-CoV resulting from the sample indexed under 031589, which sequence corresponding to the nucleotides of the positions 21406 to 25348 (SEQ ID NO: 4), with reference to the Genbank sequence AY274119.3, the plasmid, called SARS-S 1, included in the bacterial strain deposited under No. I-3020, on May 12, 2003, from the National Collection of 2862981 12 Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15; it contains a 5 'fragment of the cDNA sequence coding for the S protein of the strain of SARS-CoV resulting from the sample indexed under the number 031589, as defined above, which fragment corresponding to the nucleotides of the positions 21406 to 23454 (SEQ ID NO: 5), with reference to the Genbank sequence AY274119.3 Tor2, - the plasmid, called SARS-S2, included in the bacterial strain deposited under No. I-3019, on May 12, 2003, from the Collection National Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15; it contains a fragment 3 'of the cDNA sequence coding for the S protein of the SARS-CoV strain resulting from the sample indexed under 031589, as defined above, which fragment corresponding to the nucleotides of positions 23322 to 25348 (SEQ ID NO: 6), with reference to the Genbank sequence of access AY274119.3, the plasmid, called SARS-SE, included in the bacterial strain deposited under No. I-3126, on, November 13, 2003, from the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15; it contains the cDNA corresponding to the region between the ORF-S and the ORF-E and overlapping the ORF-E of the strain of SARS-CoV resulting from the sample indexed under the number 031589, as defined above. above, which region corresponding to the nucleotides of positions 25110 to 26244 (SEQ ID NO: 8), with reference to the Genbank n access sequence AY274119.3, the plasmid, called SARS-E, included in the bacterial strain deposited under I-3046, May 28, 2003, from the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15; it contains the cDNA sequence coding for the E protein of the SARS-CoV strain resulting from the sample indexed under the number 031589, as defined above, which sequence corresponding to the nucleotides of the positions 26082 to 26413 (SEQ ID NO: 15), with reference to Genbank accession sequence AY274119.3, - the plasmid, called SARS-M; included in the bacterial strain deposited under No. I-3047, on May 28, 2003, with the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15; it contains the cDNA sequence coding for the M protein of the strain of SARS-CoV resulting from the sample indexed under the number 031589, as defined above; Which sequencecorresponds to the nucleotides of the positions 26330 to 27098 (SEQ ID NO: 18), with reference to the Genbank n access sequence AY274119.3, the plasmid called SARS-MN, included in the bacterial strain deposited under the name I-3125, November 13, 2003, from the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15; it contains the cDNA sequence corresponding to the region between the ORF-M and the ORF-N of the strain of SARS-CoV resulting from the sample indexed under 031589 and taken from Hanoi, as defined above which sequence corresponds to the nucleotides of the positions 26977 to 28218 (SEQ ID NO: 20), with reference to the Genbank n access sequence AY274119.3, the plasmid called SARS-N, included in the bacterial strain deposited under I-3048, June 5, 2003, from the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15; it contains the cDNA coding for the N protein of the strain of SARS-CoV resulting from the sample indexed under the number 031589, as defined above, which sequence corresponding to the nucleotides of the positions 28054 to 29430 (SEQ ID NO: 38) , with reference to the Genbank sequence of access AY274119.3, the plasmid called SARS-5'NC, included in the bacterial strain deposited under No. I-3124, on November 7, 2003, from the National Collection of Cultures Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15; it contains the cDNA corresponding to the 5 'non-coding end of the genome of the strain of SARS-CoV resulting from the sample numbered under 031589, as defined above, which sequence corresponding to the nucleotides of positions 1 to 204 ( SEQ ID NO: 39), with reference to Genbank sequence AY274119.3 access, - the plasmid called SARS-3'NC, included in the bacterial strain deposited under No. I-3123 on November 7, 2003, with the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15.; it contains the cDNA sequence corresponding to the 3 'non-coding end of the genome of the strain of SARS-CoV resulting from the sample indexed under 031589, as defined above, which sequence corresponds to that located between the nucleotide in position 28933 to 29727 (SEQ ID NO: 40), with reference to the Genbank sequence of accession AY274119.3, ends with a series of nucleotides a., 2862981 14 - the expression plasmid called pIV2.3N, containing a cDNA fragment coding for a C-terminal fusion of the N protein (SEQ ID NO: 37) with a polyhistidine label, - the expression plasmid called pIV2.3Sc, containing a cDNA fragment coding for a fusion C-terminus of the fragment corresponding to positions 475 to 1193 of the amino acid sequence of protein S (SEQ ID NO: 3) with a polyhistidine tag, - the expression plasmid pIV2.3SL, containing a coding cDNA fragment for a C-terminal fusion of the corresponding fragment at positions 14 to 1193 of the amino acid sequence of protein S (SEQ ID NO: 3) with a polyhistidine tag, - the expression plasmid called pIV2.4N, containing a cDNA fragment encoding a fusion N-terminal protein N (SEQ ID NO: 3) with a polyhistidine label, - the expression plasmid called pIV2.4Sc or pIV2.4S1, containing an insert coding for an N-terminal fusion of the fragment corresponding to positions 475 at 1193 of the amino acid sequence of the protein S (SEQ ID NO: 3) with a polyhistidine tag, and - the expression plasmid called pIV2.4SL containing a cDNA fragment coding for an N-terminal fusion of the fragment corresponding to positions 14 to 1193 of the amino acid sequence of protein S (SEQ ID NO: 3) with a polyhistidine tag.

  According to an advantageous arrangement of the expression plasmid as defined above, it is included in a bacterial strain which was deposited under the number I-3117, on October 23, 2003, with the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15.

  According to another advantageous arrangement of the expression plasmid as defined above, it is included in a bacterial strain which was deposited under the number I-3118, on October 23, 2003, with the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15.

  The present invention also relates to a cDNA library characterized in that it comprises fragments as defined above, in particular amplification fragments or restriction fragments, cloned in a recombinant vector, in particular an expression vector (expression library).

  The subject of the present invention is also cells, in particular prokaryotic cells, modified with a recombinant vector as defined above.

  The recombinant vectors as defined above and the cells transformed with said expression vectors are advantageously used for the production of the corresponding proteins and peptides. The expression libraries derived from said vectors, as well as the cells transformed by said expression libraries, are advantageously used to identify the immunogenic epitopes (B and T epitopes) of the SARS-associated coronavirus proteins.

  The present invention also relates to purified or isolated proteins and peptides, characterized in that they are encoded by the polynucleotide or one of its fragments as defined above.

  According to an advantageous embodiment of the invention, said protein is selected from the group consisting of: the protein S of sequence SEQ ID NO: 3 - the protein E of sequence SEQ ID NO: 14 - the protein M of sequence SEQ ID NO: 17 - the protein N of sequence SEQ ID NO: 37 - the proteins encoded by the ORFs: ORFIa, ORF1b, ORF3, ORF4 and ORF7 to ORF11, ORF13 and ORF14 of sequence respectively, SEQ ID NO: 74, 75, 10, 12, 22, 24, 26, 28, 30, 33 and 35.

  According to an advantageous embodiment of the invention, said peptide is selected from the group consisting of: a) the peptides corresponding to the positions 14 to 1193 and 475 to 1193 of the amino acid sequence of the protein S, b) the peptides corresponding to positions 2 to 14 (SEQ ID NO: 69) and 100 to 221 of the amino acid sequence of protein M; these peptides correspond respectively to the ectodomain and endodomain of the M protein, and c) the peptides corresponding to positions 1 to 12 (SEQ ID NO: 70) and 53 to 76 (SEQ ID NO: 71) of the amino acid sequence of protein E; these peptides correspond respectively to the ectodomain and the C-terminal end of the E protein, and d) the peptides of 5 to 50 consecutive amino acids, preferably from 10 to 30 amino acids, included or partially overlapping or totally the sequence of the peptides as defined in a), b) or c).

  The subject of the present invention is also a peptide characterized in that it has a sequence of 7 to 50 amino acids, including an amino acid residue selected from the group consisting of: alanine located at position 2552 of the sequence in amino acids of the protein encoded by ORF 1 a.

  the serine located at position 577 of the amino acid sequence of the S protein of the SARS-CoV strain as defined above, the glycine at position 11 of the amino acid sequence of the protein encoded by the ORF3 of the SARS-CoV strain as defined above, the serine at position 154 of the amino acid sequence of the M protein of the SARS-CoV strain as defined above.

  The subject of the present invention is also an antibody or a polyclonal or monoclonal antibody fragment, obtainable by immunization of an animal with a recombinant vector as defined above, a cDNA library as defined above or a protein or a peptide as defined above, characterized in that it binds with at least one of the proteins encoded by the SARS-CoV as defined above.

  The invention encompasses polyclonal antibodies, monoclonal antibodies, chimeric antibodies such as humanized antibodies, and fragments thereof (Fab, Fv, scFv).

  For the purposes of the present invention, the term "chimeric antibody" means, relative to an antibody of a particular animal species or of a particular class of antibody, an antibody comprising all or part of a heavy chain and / or a a light chain of an antibody of another animal species or another class of antibodies.

  For the purposes of the present invention, the term "humanized antibody" is intended to mean a human immunoglobulin in which the residues of the CDRs (Complementary-Determining Regions) which form the antigen-binding site are replaced by those of a non-monoclonal antibody. human having the specificity, affinity or activity sought. In comparison with non-human antibodies, humanized antibodies are less immunogenic and have a prolonged half-life in humans because they have only a small proportion of non-human sequences, since almost all The residues of the FR regions (Framework) and the constant region (Fc) of these antibodies are those of a consensus sequence of human immunoglobulins.

  The present invention also relates to a protein chip, characterized in that it comprises a protein, a peptide or an antibody as defined above.

  The protein chips according to the invention are prepared by conventional methods, known in themselves. Suitable carriers on which proteins may be immobilized include those made of plastic or glass, especially in the form of microplates.

  The present invention also relates to reagents derived from the strain isolated from SARS-associated coronavirus derived from the sample numbered 031589, useful for the study and diagnosis of infection caused by a SARS-associated coronavirus, which The reagents are selected from the group consisting of: (a) a primer pair, a probe or a microarray as defined above, (b) a recombinant vector or a modified cell as defined above, c) an isolated strain of coronavirus or a polynucleotide as defined above, (d) a protein or a peptide as defined above, (e) an antibody or antibody fragment as defined above, and (f) a protein chip as defined above.

  These different reagents are prepared and used according to the standard techniques of molecular biology and immunology, following standard protocols such as those described in Current Protocols in Molecular Biology 2862981 (Frederick M. AUSUBEL, 2000, Wiley and Son Inc., Library of Congress, USA), in Current Protocols in Immunology (John E. Cologan, 2000, Wiley and Son Inc. Library of Congress, USA) and in Antibodies: A Laboratory Manual (E. Howell and D Lane, Cold Spring Harbor Laboratory , 1988).

  The nucleic acid fragments according to the invention are prepared and used according to the conventional techniques as defined above. The peptides and proteins according to the invention are prepared by recombinant DNA techniques known to those skilled in the art, in particular using the recombinant vectors as defined above. Alternatively, the peptides according to the invention can be prepared by conventional solid or liquid phase synthesis techniques known to those skilled in the art.

  The polyclonal antibodies are prepared by immunizing an appropriate animal with a protein or a peptide as defined above, optionally coupled to KLH or albumin and / or combined with a suitable adjuvant such as Freund's adjuvant. (complete or incomplete) or alumina hydroxide; after obtaining a satisfactory antibody titre, the antibodies are harvested by taking the serum of the immunized animals and enriched with IgG by precipitation, according to standard techniques, then the SARS-CoV protein-specific IgGs are optionally purified by chromatography of affinity on an appropriate column to which said peptide or said protein, as defined above, is attached so as to obtain a monospecific IgG preparation.

  The monoclonal antibodies are produced from hybridomas obtained by fusion of B lymphocytes of an animal immunized with a protein or a peptide as defined above with myelomas, according to the technique of Ki hier and Milstein (Nature, 1975, 256, 495-497); the hybridomas are cultured in vitro, in particular in fermentors or produced in vivo, in the form of ascites; alternatively, said monoclonal antibodies are produced by genetic engineering as described in US Pat. No. 4,816,567.

  Humanized antibodies are produced by general methods such as those described in International Application WO 98/45332.

  Antibody fragments are produced from cloned VH and VL regions from hybridoma or spleen cell mRNAs of an immunized mouse; for example, Fv, scFv or Fab fragments are expressed on the surface of filamentous phages according to the technique of Winter and Milstein (Nature, 1991, 349, 293-299); after several selection steps, the antigen-specific antibody fragments are isolated and expressed in a suitable expression system, by standard techniques of cloning and expression of recombinant DNA.

  The antibodies or their fragments as defined above are purified by standard techniques known to those skilled in the art, such as affinity chromatography.

  The present invention further relates to the use of a product selected from the group consisting of: a pair of primers, a probe, a DNA chip, a recombinant vector, a modified cell, an isolated strain of coronavirus, a polynucleotide, a protein or a peptide, an antibody or an antibody fragment, and a protein chip as defined above, for the preparation of a reagent for detecting and possibly genotyping / serotyping a coronavirus associated with SARS.

  The proteins and peptides according to the invention, which are capable of being recognized and / or of inducing the production of antibodies specific for SARS-associated coronavirus, are useful for the diagnosis of infection by such a coronavirus; the infection is detected by a suitable technique - in particular EIA, ELISA, RIA, immunofluorescence-, from a biological sample taken from an individual likely to be infected.

  According to an advantageous arrangement of said use, said proteins are selected from the group consisting of the S, E, M and / or N proteins and the peptides as defined above.

  The S, E, M and / or N proteins and the peptides derived from these proteins as defined above, for example the N protein, are used for the indirect diagnosis of a coronavirus infection associated with SARS (serological diagnosis; detection of SARS-CoV specific antibodies), in particular by an immunoenzymatic method (ELISA).

  The antibodies and the antibody fragments according to the invention, in particular those directed against the proteins S, E, M and / or N and the peptides derived as defined above, are useful for the direct diagnosis of a human infection. SARS-associated navirus coronary agent; the detection of SARS-CoV protein (s) is carried out by an appropriate technique, in particular EIA, ELISA, RIA, immunofluorescence from a biological sample taken from an individual susceptible to infection.

  The present invention also relates to a method for detecting a SARS-associated coronavirus from a biological sample, which method is characterized in that it comprises at least: (a) contacting said biological sample with at least one antibody or an antibody fragment, a protein, a peptide or a protein or peptide chip or filter as defined above, and (b) revealing by any appropriate means the complexes antigen-antibodies formed in (a), for example by EIA, ELISA, RIA, or by immunofluorescence.

  According to an advantageous embodiment of said method, step (a) comprises: (a1) bringing said biological sample into contact with at least a first antibody or an antibody fragment which is fixed on a suitable support, in particular a microplate, (a2) washing the solid phase, and (a3) adding at least a second antibody or an antibody fragment, different from the first, said antibody or antibody fragment being optionally labeled with appropriate.

  This method which makes it possible to capture the viral particles present in the biological sample is also called the immunocapture method. For example: step (a1) is carried out with at least a first monoclonal or polyclonal antibody or a fragment thereof, directed against the protein S, M, and / or E, and / or a peptide corresponding to the ectodomain of one of these proteins (peptides M2-14 or El-12) step (a3) is carried out with at least one antibody or an antibody fragment directed against another epitope of the same protein or preferably against another protein, preferably against an internal protein such as nucleoprotein N or the endodomain of the protein E or M, even more preferably 2862981 21 it is antibodies or fragments of antibodies directed against the N protein which is very abundant in the viral particle; when an antibody or an antibody fragment directed against an internal protein (N) or against the endodomain of the E or M proteins is used, said antibody is incubated in the presence of detergent, such as Tween 20 for example, with concentrations of the order of 0.1%.

  step (b) of revealing the antigen-antibody complexes formed is carried out, either directly with the aid of a second antibody labeled for example with biotin or an appropriate enzyme such as peroxidase or alkaline phosphatase, or indirectly using an anti-immunoglobulin serum labeled as above. The complexes thus formed are revealed using a suitable substrate.

  The present invention further relates to a kit for detecting a coronavirus associated with SARS, characterized in that it comprises at least one reagent selected from the group consisting of: a pair of primers, a probe, a chip to DNA or RNA, a recombinant vector, a modified cell, an isolated strain of coronavirus, a polynucleotide, a protein or a peptide, an antibody, and a protein chip as defined above.

  The present invention further relates to an immunogenic composition, characterized in that it comprises at least one product selected from the group consisting of: a) a protein or a peptide as defined above, b) a polynucleotide of DNA or RNA type or one of its representative fragments as defined above, of sequence chosen from: (i) the sequence SEQ ID NO: 1 or its RNA equivalent (ii) the sequence hybridizing under conditions of strong stringency with the sequence SEQ ID NO: 1, (iii) the sequence complementary to the sequence SEQ ID NO: 1 or the hybridizing sequence under conditions of high stringency with the sequence SEQ ID NO: 1, (iv) the nucleotide sequence a representative fragment of the polynucleotide as defined in (i), (ii) or (iii), (v) the sequence as defined in (i), (ii), (iii) or (iv), modified, and 2862981 c) a recombinant expression vector comprising a polynucleotide as defined in b) and d) a cDNA library as defined above, said immunogenic composition being capable of inducing a protective humoral or cellular immunity specific for the SARS-associated coronavirus, in particular the production of an antibody directed against a specific epitope of the coronavirus associated with SARS.

  The proteins and peptides as defined above, in particular the S, M, E and / or N proteins and the derived peptides, as well as the nucleic acid molecules (DNA or RNA) coding for said proteins or said peptides, are good vaccine candidates and can be used in immunogenic compositions for the production of a vaccine against SARS-associated coronavirus.

  According to an advantageous embodiment of the compositions according to the invention, they also contain at least one pharmaceutically acceptable vehicle and optionally carrier substances and / or adjuvants.

  The pharmaceutically acceptable vehicles, the carrier substances and the adjuvants are those conventionally used.

  The adjuvants are advantageously selected from the group consisting of oily emulsions, saponin, mineral substances, bacterial extracts, alumina hydroxide and squalene.

  The carrier substances are advantageously selected from the group consisting of unilamellar liposomes, multilamellar liposomes, saponin micelles or solid microspheres of saccharide or auriferous nature.

  The compositions according to the invention are administered by the general route, in particular intramuscularly or subcutaneously, or by the local route, in particular the nasal route (aerosol).

  The present invention also relates to the use of an isolated or purified protein or peptide having a sequence selected from the group consisting of the sequences SEQ ID NO: 3, 10, 12, 14, 17, 22, 24 , 26, 28, 30, 33, 35, 37, 69, 70, 71, 74 and 75 to form an immune complex with an antibody specifically directed against a SARS-associated coronavirus epitope.

  The present invention also relates to an immune complex formed of an isolated or purified protein or peptide having a sequence selected from the group consisting of the sequences SEQ ID NO: 3, 10, 12, 14, 17, 22, 24, 26, 28, 30, 33, 35, 37, 69, 70, 71, 74 and 75, and an antibody specifically directed against a SARS-associated coronavirus epitope.

  The present invention also relates to the use of an isolated or purified protein or peptide having a sequence selected from the group consisting of the sequences SEQ ID NO: 3, 10, 12, 14, 17, 22, 24 , 26, 28, 30, 33, 35, 37, 69, 70, 71, 74 and 75 to induce the production of an antibody capable of specifically recognizing a SARS-associated coronavirus epitope.

  The present invention also relates to the use of an isolated or purified polynucleotide having a sequence selected from the group consisting of the sequences SEQ ID NO: 1, 2, 4, 7, 8, 13, 15, 16, 18, 19, 20, 31, 36 and 38 for inducing the production of an antibody directed against the protein encoded by said polynucleotide and capable of specifically recognizing a SARS-associated coronavirus epitope. In addition to the foregoing, the invention further comprises other arrangements, which will emerge from the description which follows, which refers to examples of implementation of the polynucleotide representing the genome of the SARS-CoV strain from the sample numbered 031589, and fragments of cDNA derivatives Objects of the present invention, as well as in Table I showing the sequence listing: Table I: Sequence list Sequence number Sequence number of the identification number cDNA in depot at CNCM refers to the corresponding Genbank plasmid AY274119.3 SEQ ID NO: 1 genome of the - strain resulting from the sampling SEQ ID NO: 2 ORF-S * 21406-25348 - SEQ ID NO: 3 Protein S - SEQ ID NO: 4 ORF-S ** 21406-25348 1-3059 SEQ ID NO: 5 fragment Sa 2140623454 I-3020 2862981 24 SEQ ID NO: 6 fragment Sb 23322-25348 I-3019 SEQ ID NO: 7 ORF-3 + ORF-4 * 25110-26244 - SEQ ID NO: 8 ORF-3 + ORF-4 ** 25110-26244 I-3126 SEQ ID NO: 9 ORF3 - - SEQ ID NO: 10 ORF-3 Protein - - SEQ ID NO: 11 ORF4 - - SEQ ID NO: 12 ORF-4 Protein - - SEQ ID NO: 13 ORF-E * 26082-26413 - SEQ ID NO: 14 Protein E - - SEQ ID NO: 15 ORF-E ** 26082-26413 I-3046 SEQ ID NO: 16 ORF-M * 26330-27098-SEQ ID NO: 17 Protein M-SEQ ID NO: 18 ORF-M ** 26330-27098 I-3047 SEQ ID NO: 19 ORF7-11 * 26977-28218 - SEQ ID NO: 20 ORF7 to 11 ** 26977-28218 1-3125 SEQ ID NO: 21 ORF7 - - SEQ ID NO: 22 ORF7 protein - - SEQ ID NO: 23 ORF8 - - SEQ ID NO: 24 ORF8 protein - - SEQ ID NO: 25 ORF9 - - SEQ ID NO: 26 ORF9 protein - - SEQ ID NO : 27 ORF10 - - SEQ ID NO: 28 ORF10 protein - - SEQ ID NO: 29 ORF11 - - SEQ ID NO: 30 ORF11 protein - - SEQ ID NO: 31 OrF1 ab 265-21485 - SEQ ID NO: 32 ORF13 28130- 28426 - SEQ ID NO: 33 ORF13 Protein - - SEQ ID NO: 34 ORF14 - - SEQ ID NO: 35 ORF14 Protein 28583-28795 - SEQ ID NO: 36 ORF-N * 28054-29430 SEQ ID NO: 37 N Protein - SEQ ID NO: 38 ORF-N ** 28054-29430 I-3048 SEQ ID NO: 39 5'non-coding ** 1- 204 I-3124 SEQ ID NO: 40 3'non-coding ** 28933-29727 I-3123 SEQ ID NO: 41 ORF1 ab 30-500 - Fragment LO SEQ ID NO: 42 Fragment L1 211-2260 - SEQ ID NO: 43 Fragment L2 2136-4187 - SEQ ID NO: 44 Fragment L3 3892-5344 - SEQ ID NO: 45 Fragment Lob 4932-6043 - SEQ ID NO: 46 Fragment L4 5305-7318 - SEQ ID NO: 47 Fragment L5 7275-9176 - SEQ ID NO: 48 Fragment L6 9032-11086 - SEQ ID NO: 49 Fragment L7 10298-12982 - SEQ ID NO: 50 Fragment L8 12815-14854 2862981 SEQ ID NO: 51 Fragment L9 14745-16646 - SEQ ID NO: 52 Fragment L10 16514-18590 - SEQ ID NO: 53 Fragment L11 18500-20602 - SEQ I D NO: 54 Fragment L12 20319-22224 - SEQ ID NO: 55 Primer N sense - - SEQ ID NO: 56 Primer N - - antisense SEQ ID NO: 57 Primer Sc sense - - SEQ ID NO: 58 Primer SL sense - - SEQ ID NO: 59 Primer Sc and SL - - antisense SEQ ID NO: 60 Primer sense 28507-28522 - series 1 SEQ ID NO: 61 Primer antisense 28774-28759 series 1 SEQ ID NO: 62 Primer sense 28375-28390 - series 2 SEQ ID NO: 63 28702-28687 Reverse Primer - Series 2 SEQ ID NO: 64 1 / Series 1 Probe 28561-28586 - SEQ ID NO: 65 Probe 2 / Series 1 28588-28608 - SEQ ID NO: 66 Probe 1 / Serial 2 28541-28563 - SEQ ID NO: 67 Probe 2 / series 2 28565-28589 SEQ ID NO: 68 Primer anchor _ 14T SEQ ID NO: 69 Peptide M2-14 - - SEQ ID NO: 70 Peptide E1-12 - - SEQ ID NO: 71 Peptide E53-76 - - SEQ ID NO: 72 5'non-coding * 1-204 - SEQ ID NO: 73 3'non-coding * 28933-29727 - SEQ ID NO: 74 Protein ORF1a - - SEQ ID NO: 75 Protein ORF1b - - SEQ ID NO: 76-139 Primers * amplification PCR product (amplicon) ** insert cloned into the plasmid deposited at the CNCM as well as to the accompanying drawings, in which: FIG. 1 illustrates the Western blot analysis of the in vitro expression of recombinant N, Sc and SL proteins from the pIVEX expression vectors. Lane 1: pIV2.3N. Track 2: pIV2.3Sc. Track 3: pIV2. 3SL. Lane 4: pIV2.4N. Track 5: pIV2.4SI or pIV2.4Sc. Track 6: pIV2.4SL. Expression of the GFP protein expressed from the same vector is used as a control.

  FIG. 2 illustrates the denaturing polyacrylamide gel electrophoresis analysis (SDS-PAGE) and coomassie blue staining, of the in vivo expression of the N protein from the pIVEX expression vectors. . The E. coli strain BL21 (DE3) pDIA17 transformed by the recombinant pIVEX vectors is cultured at 30 ° C. in LB medium, in the presence or absence of inducer (1 mM IPTG). Track 1: pIV2.3N Track 2: pIV2.4N. FIG. 3 illustrates the gel electrophoresis analysis of

  polyacrylamide under denaturing conditions (SDS-PAGE) and staining with Coomassie blue, the in vivo expression of the SL and Sc polypeptides from the pIVEX expression vectors. The E. coli strain BL21 (DE3) pDIA17 transformed with the recombinant pIVEX vectors is cultured at 30 ° C. in LB medium, in the presence or absence of inducer (IPTG ImM). Track 1: pIV2.3Sc Track 2: pIV2.3SL. Track 3: pIV2.4S1 Track 4: pIV2. 4SL.

  FIG. 4 illustrates the antigenic activity of the recombinant N, SL and Sc proteins produced in E. coli strain BL21 (DE3) pDIA17 transformed by the recombinant pIVEX vectors. A: electrophoresis (SDS-PAGE) bacterial lysates.B and C: Western blot with sera, from the same patient infected with SARS-CoV, taken respectively 8 days (B: M12 serum) and 29 days - (C: M13 serum) after the onset of SARS symptoms. Lane 1: pIV2.3N. Track 2: pIV2.4N. Lane 3: pIV2.3Sc. Lane 4: pIV2.4 Si. Lane 5: pIV2.3SL. Lane 6: pIV2.4SL FIG. 5 illustrates the purification on a Ni-NTA agarose column of the recombinant N protein produced in E. coli strain BL21 (DE3) pDIA17 from the pIV2.3N vector. Lane 1: Total bacterial extract. Lane 2: Soluble extract. Lane 3: Insoluble extract. Lane 4: Extract deposited on the Ni-NTA column. Lane 5: Non-retained proteins. Track 6: Peak 1 Fractions. Track 7: Peak 2 Fractions.

  FIG. 6 illustrates the purification of the recombinant Sc protein from the inclusion bodies produced in the E. coli strain BL21 (DE3) pDIA17 transformed with pIV2.4S1.A. Treatment with Triton X-100 (2%): Track 1: Total bacterial extract. Lane 2: Soluble extract. Lane 3: Insoluble extract. Lane 4: Supernatant after treatment with Triton X-100 (2%). Lane 5 and 6: Pellet after treatment with Triton X-100 (2%) B: Treatment with 4M, 5M, 6M and 7M urea of soluble and insolubic extracts.

  FIG. 7 represents the immunoblotting performed using a lysate of cells infected with SARS-CoV and a serum of a patient suffering from atypical pneumonitis.

  FIG. 8 represents immunoblots made using a lysate of cells infected with SARS-CoV and antisera of rabbits specific for nucleoprotein N (A) and spike protein S (B). I.S .: immune serum. p.i .: pre-immune serum. Anti-N antiserum was used at 1/50000 and antiserum anti-S at 1/10000.

  FIG. 9 illustrates the ELISA reactivity of rabbit-specific monoclonal polyclonal sera directed against the N protein or the short fragment of the S (Sc) protein, with respect to the corresponding recombinant proteins used for immunization. A: rabbits P13097, P13081, and P13031 immunized with purified recombinant N protein. B: rabbits P11135, P13042, and P14001 immunized with an inclusion body preparation corresponding to the short fragment of the protein S (Sc). I. S. : immune serum p.i .: pre-immune serum.

  FIG. 10 illustrates the ELISA reactivity of the purified recombinant N protein with respect to serum of patients suffering from atypical pneumonia caused by SARS-CoV. Figure 10a: ELISA plates prepared with N protein at the concentration of 4 11g / ml and 2 g / ml. Figure 10b: ELISA plate prepared with N protein at a concentration of 1 μg / ml. Serums designated A, B, D, E, F, G, H correspond to those of Table IV.

  FIG. 11 illustrates the amplification by RT-PCR of decreasing amounts of synthetic SARS-CoV N gene RNA (107 to 1 copy), using primer pairs n 1 (N / + / 28507). , N / - / 28774) (A) and n 2 (N / + / 28375, N / - / 28702) (B). T: amplification carried out in the absence of RNA. MW: DNA marker.

  FIG. 12 illustrates the real-time RT-PCR amplification of synthetic SARS-CoV N gene RNA: decreasing amounts of synthetic RNA in replicate (folds, lanes 16 to 29) as well as 1 / 20x10 diluted viral RNA (lane 32) were amplified by RT-PCR in real time using the "Light Cycler RNA Amplification Kit Hybridization Probes" kit and n 2 series of primers and probes under the conditions described in Example 7.

  FIG. 13.1 to 13.70 represents the restriction map of the sequence SEQ ID NO: 1 corresponding to the DNA equivalent of the genome of the strain of SARS-CoV resulting from the sample indexed under the number 031589.

  It should be understood, however, that these examples are given solely by way of illustration of the object of the invention, of which they in no way constitute a limitation.

  EXAMPLE 1 Cloning and Sequencing of the Genome of the SARS-CoV Strain Derived from the Collection Listed Under the Number 031589 RNA from the strain of SARS-CoV was extracted from the bronchoalveolar lavage sample under the number 031589, carried out on a patient of the French hospital of Hanoi (Vietnam) with SARS.

  Isolated RNA was used as a template to amplify cDNAs corresponding to different open reading frames of the genome (ORF 1a, ORF 1b, ORF-5, ORF-E, ORF-M, ORF-N (including ORF-13 and ORF-14), ORF3, ORF4, ORF7 to ORF11), and the 5 'and 3' non-coding ends. The sequences of primers and probes used for amplification / detection were defined according to the available nucleotide sequence of SARS-CoV.

  In the following the primers and probes are identified by: the letter S, followed by a letter which indicates the corresponding region of the genome (L for the end including ORF la and ORF lb; S, M and N for the ORF-S, ORF-M, ORF-N, SE and MN for the corresponding intergenic regions), then optionally Fn, Rn, with n included between 1 and 6 corresponding to the primers used for the nested or nested PCR (IF pair + RI for the first amplification, pair F2 + R2 for the second amplification, etc ...), then / + / or / / corresponding to a sense or antisense primer and finally positions of the primers with reference to the sequence Genbank AY27411 .3; for S and N sense and antisense primers and other sense primers only, when a single position is indicated it corresponds to that of the 5 'end of a probe or primer of about 20 bases; for antisense primers other than S and N primers, when a single position is indicated it corresponds to that of the 3 'end of a probe or primer of about 20 bases.

  The amplification products thus generated were sequenced using specific primers in order to determine the complete genome sequence of the strain SARS-CoV resulting from the sample indexed under the number 031589. These amplification products, with the exception of those corresponding to ORF 1a and ORF 1b, were then cloned into expression vectors to produce the corresponding viral proteins and antibodies directed against these proteins, in particular by DNA-based immunization.

  1. RNA extraction The RNAs were extracted using the Qlamp viral RNA mini extraction kit (QIAGEN) following the manufacturer's recommendations. More precisely: 140 d of the sample and 560 l of AVL buffer were mixed vigorously for 15 seconds, incubated for 10 min at room temperature and then briefly centrifuged at maximum speed. 560 l of 100% ethanol was added to the supernatant and the resulting mixture was stirred very vigorously for 15 sec. 630 of the mixture were then deposited on the column.

  The column was placed on a 2 ml tube, centrifuged for 1 min at 8000 rpm, then the rest of the previous mixture was deposited on the same column, centrifuged again, 1 min at 8000 rpm and the column was transferred to a 2 ml tube clean. Then, 500 μl of AW1 buffer was added to the column, then the column was centrifuged for 1 min at 8000 rpm and the eluate was removed. 500 l of AW2 buffer was added to the column which was then centrifuged for 3 min at 14000 rpm and transferred to a 1.5 ml tube. Finally, 60 l of AVE buffer was added to the column which was incubated for 1 to 2 min at room temperature and then centrifuged for 1 min at 8000 rpm. The eluate corresponding to the purified RNA was recovered and frozen at -20 ° C.

  2. Amplification, sequencing and cloning of cDNAs 2.1) cDNA encoding protein S The RNAs extracted from the sample were reverse transcribed using random sequence hexameric oligonucleotides (pdN6) to produce cDNA fragments.

  The sequence encoding S-glycoprotein of SARS-CoV was amplified as two overlapping DNA fragments: 5 'fragment (SARS-Sa, SEQ ID NO: 5) and 3' fragment (SARS-Sb, SEQ ID NO: 6), performing two successive amplifications using nested primers. The amplicons thus obtained 2862981 were sequenced, cloned into the plasmid vector PCR 2.1-TOPOTM (1N VITROGEN), and the sequence of the cloned cDNAs was determined.

  a) Cloning and Sequencing of the Sa and Sb Fragments a) synthesis of the cDNA The reaction mixture containing: RNA (5 μl), H2O ppi (3.5 l), 5X reverse transcriptase buffer (4 μl), 5 mM dNTP (2 μl), pdN6 100 μg / ml (4 μl), RNasin 40 μlul (0.5 μl) and reverse transcriptase AMV-RT, Ullul, PROMEGA (1 μl) was incubated in a thermal cycler under the following conditions: 45 min at 42 ° C., 15 min at 55 ° C., 5 min at 95 ° C., then the cDNA obtained was maintained at +4 ° C.

  a2) first.amplification.PÇR The 5 'and 3' ends of the S gene were amplified respectively with primer pairs S / Fl / + / 21350-21372 and S / R1 / - / 23518-23498, S / F3 / + / 23258-23277 and SIR3 / - / 25382-25363. The 50 l reaction mixture containing: cDNA (2 1.1), 50 M primers (0.5 l), 10 X buffer (5 μl), 5 mM dNTP (2 μl), Taq Expand High Fidelity, Roche (0.75). pl) and H 2 O (39.75 μl) was amplified in a thermocycler under the following conditions: an initial denaturation step at 94 ° C for 2 min was followed by 40 cycles including: a denaturation step at 94 ° C for 30 min. dry, a hybridization step at 55 ° C. for 30 sec and then an extension step at 72 ° C. for 2 min 30 sec, with 10 sec of additional elongation at each cycle, followed by a final step of elongation at 72 ° C. C for 5 min. a3) second PCR amplification The products of the first PCR amplification (5 'and 3' amplicons) underwent a second PCR amplification step (nested PCR) under conditions identical to those of the first amplification, with the primer pairs S / F2 / + / 21406-21426 and S / R2 / - / 23454-23435, and S / F4 / + / 23322-23341 and S / R4 / -125348-25329 respectively for the amplicon 5 'and the amplicon 3 '.

  a4) cloning and. Sequencing of Sa and Sb fragments. The amplicons Sa (5 'end) and Sb (3' end) thus obtained were purified using the QlAquick PCR purification kit (QIAGEN), following the manufacturer's recommendations, and then they were cloned in the vector PCR2.1-TOPO (Invitrogen kit), to give the plasmids called SARS-S1 and SARS-S2.

  The DNA of clones Sa and Sb was isolated and the corresponding insert was sequenced using the Big Dye Kit, Applied Biosystem and the universal primers M13 forward and M13 reverse, as well as primers: S / S / + / 21867, S / S / + 122353, S / S / + / 22811, S / S / + / 23754, S / S / + / 24207, S / S / + 124699, SIS / + / 24348, SISI - / 24209, S / S / - / 23630, SIS / - / 23038, SIS // 22454, SIS / - / 21815, S / S / - / 24784, S / S / + / 21556, SIS / + / 23130 and SISI + / 24465, following the manufacturer's instructions; the sequences of fragments Sa and Sb thus obtained correspond to the sequences SEQ ID NO: 5 and SEQ ID NO: 6 in the attached sequence listing.

  The plasmid, called SARS-S1 was deposited under No. I-3020, on May 12, 2003, with the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15; it contains a 5 'fragment of the S gene sequence of the SARS-CoV strain resulting from the sample indexed under the number 031589, as defined above, which fragment designated Sa corresponding to the nucleotides of the positions 21406 to 23454 (SEQ ID NO: 5), with reference to Genbank sequence AY274119.3 Tort.

  The plasmid, called TOP10F'-SARS-S2 was deposited under the I-3019, on May 12, 2003, at the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15; it contains a fragment 3 'of the S gene sequence of the strain of SARS-CoV resulting from the sample listed under No. 031589, as defined above, which fragment called Sb corresponding to the nucleotides of positions 23322 to 25348 ( SEQ ID NO: 6), with reference to the Genbank sequence of access AY274119.3.

  b) Cloning and sequencing of the complete cDNA (4 kb SARAS-S clone) The complete S-cDNA was obtained from the above SARS-S1 and SARS-S2 clones, as follows: 1) a reaction PCR amplification was performed on a SARS-S2 clone in the presence of the aforementioned primer S / R4 / - / 25348-25329 and the primer S / S / + / 24696-24715: a 633 bp amplicon 2) another PCR amplification reaction was performed on another SARS-S2 clone, in the presence of the abovementioned primers S / F4 / + / 23322-23341 and S / S / - / 24803-24784: a 1481 bp amplicon was obtained, 2862981 The amplification reaction was carried out under the conditions as defined above for the amplification of fragments Sa and Sb, except that 30 cycles of amplifications comprising a step denaturation at 94 C for 20 sec and an elongation step at 72 C for 2 min 30 sec were performed.

  3) the 2 amplicons (633 bp and 1481 bp) were purified under the conditions as defined above for the fragments Sa and Sb.

  4) another PCR amplification reaction using the primers S / F4 / + / 23322-23341 and SIR4 / - / 25348-25329 above, was carried out on the purified amplicons obtained in 3). The amplification reaction was carried out under the conditions as defined above for the amplification of fragments Sa and Sb, with the exception that 30 cycles of amplifications were carried out.

  The 2026 bp amplicon thus obtained was purified, cloned into the PCR2.1-TOPO vector and then sequenced as above, using the primers as defined above for the Sa and Sb fragments. The clone thus obtained was called clone 3 '.

  5) The previously obtained SARS-S1 clone and the clone 3 'were digested with EcoR I, the bands of approximately 2kb thus obtained were purified on gel then amplified by PCR with primers S / F2 / + / 21406-21426 and S / R4 / 25348-25329 supra. The amplification reaction was carried out under the conditions as defined above for the amplification of fragments Sa and Sb, with the exception that 30 cycles of amplifications were carried out. The amplicon of about 4 kb was purified and sequenced. It was then cloned into the PCR2.1-TOPO vector to give the plasmid, called SARS-S, and the insert contained in this plasmid was sequenced as above, using the primers as defined above. above for fragments Sa and Sb. The cDNA sequences of the insert and of the amplicon encoding the S protein correspond respectively to the sequences SEQ ID NO: 4 and SEQ ID NO: 2 in the attached sequence listing, they encode the protein S (SEQ ID NO: 3).

  The sequence of the amplicon corresponding to the cDNA coding for the S protein of the SARS-CoV strain derived from the sample # 031589 has the following two mutations with respect to the corresponding sequences of respectively the Tor2 and Urbani isolates, the positions of the mutations being indicated with reference to the complete genome sequence of the Tor2 isolate (Genbank AY274119.3): g / t at position 23220; the alanine codon (gct) at position 577 of the amino acid sequence of the Tor2 protein S is replaced by a serine codon (tct), - c / t at position 24872: this mutation does not modify the amino acid sequence of the S protein, and the plasmid, called SARS-S, was deposited under the n I-3059, on June 20, 2003, with the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15; it contains the cDNA sequence coding for the S protein of the strain of SARS-CoV resulting from the sample indexed under 031589, which sequence corresponding to the nucleotides of the positions 21406 to 25348 (SEQ ID NO: 4), with reference to the Genbank sequence AY274119. 3.

  2.2) cDNAs Encoding M and E Proteins RNAs from sampling 031589, extracted as above, were subjected to an associated reverse transcription, during the same step (Titan One Step RT-PCR kit, Roche), to a PCR amplification reaction, using primer pairs: - S / E / Fl / + / 26051-26070 and S / E / R1 / - / 26455-26436 to amplify the ORF-E and S / M / F1 / + / 26225-26244 and S / M / R1 / - / 27148-27129 for amplifying the ORF-M.

  A first reaction mixture containing: 8.6 l of H2Oppi, 1 l of dNTPs (5 mM), 0.2 l of each of the primers (50 M), 1.25 l of DTT (100 mM) and 0.25 l of RNAsin (40UU 1) was combined with a second reaction mixture containing: 1 l RNA, 7 l of H2Oppi, 5 l of 5X RTPCR buffer and 0.5 l of enzyme mixture and the combined mixtures were incubated in a thermocycler under the following conditions: 30 min at 42 ° C., 10 min at 55 ° C., 2 min at 94 ° C. followed by 40 cycles comprising a denaturation step at 94 ° C. for 10 sec, a hybridization step at 55 ° C. for 30 sec and an elongation step at 68 C for 45 sec, with 3 sec increment per cycle and finally a step of terminal elongation at 68 C for 7 min. The amplification products thus obtained (amplicons M and E) underwent a second PCR amplification (nested PCR) using the Expand High-Fi Kit, Roche), using the pairs of primers: - S / E / F2 / + / 26082-26101 and S / E / R2 / -126413-26394 for the amplicon E, and S / M / F2 / + / 26330-26350 and SIM / R2 / - / 27098-27078 for the amplicon M. The reaction mixture containing: 2 111 of the product of the first PCR, 39.25 111 of H2Oppi, 511l of 10X buffer containing MgCl2, 2 l of dNTP (5mM), 0.51l of each of primers (50 μM) and 0.75 μl of enzyme mixture were incubated in a thermocycler under the following conditions: a denaturation step at 94 ° C for 2 min was followed by 30 cycles including a denaturation step at 94 ° C for 15 sec, a hybridization step at 60 C for 30 sec and an elongation step at 72 C for 45 sec, with 3 sec increment per cycle, and finally a step of terminal elongation at 72 C for 7 min . The amplification products obtained corresponding to the cDNAs encoding the E and M proteins were sequenced as above, using the primers: S / E / F2 / + / 26082 and S / E / R2 / - / 26394 , S / M / F2 / + / 26330, S / M / R2 / - / 27078 and S / MI + / 26636-26655 and S / M / - / 26567-26548 primers. They were then cloned, as above, to give the plasmids called SARS-E and SARS-M. The DNA of these clones was then isolated and sequenced with the aid of the universal primers M13 forward and M13 reverse as well as primers S / M / + / 26636 and S / M / -126548 mentioned above.

  The sequence of the amplicon representing the cDNA coding for the protein E (SEQ ID NO: 13) of the strain SARS-CoV from the sample no. 031589 does not have differences with respect to the corresponding sequences of the isolates AY274119.3- Tor2 and AY278741-Urbani. The sequence of the protein E of the SARS-CoV strain 031589 corresponds to the sequence SEQ ID NO: 14 in the attached sequence listing.

  The plasmid, called SARS-E was deposited under n I-3046, on May 28, 2003, with the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15; it contains the cDNA sequence coding for the E protein of the strain of SARS-CoV resulting from the sample indexed under the number 031589, as defined above, which sequence corresponding to the 2862981 nucleotides of the positions 26082 to 26413 (SEQ ID NO: 15), referring to the Genbank sequence of access AY274119.3.

  The sequence of the DNA-encoding amplicon encoding the M (SEQ ID NO: 16) of the SARS-CoV strain from Collection No. 031589 does not differ from the corresponding sequence of the AY274119.3- Tor2. On the other hand, in position 26857, isolate AY278741-Urbani contains a c and the sequence of the strain of SARS-CoV resulting from the sample listed under No. 031589 a t. This mutation results in a modification of the amino acid sequence of the corresponding protein: in position 154, a proline (AY278741-Urbani) is changed to serine in the strain of SARS-CoV resulting from the sample indexed under the number 031589. The sequence of the M protein of the SARS-CoV strain resulting from the sample indexed under 031589 corresponds to the sequence SEQ ID NO: 17 in the attached sequence listing.

  The plasmid, called SARS-M was deposited under No. I-3047, on May 28, 2003, from the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15; it contains the cDNA sequence coding for the M protein of the strain of SARS-CoV resulting from the sample indexed under the number 031589, as defined above; which sequence corresponds to the nucleotides of the positions 26330 to 27098 (SEQ ID NO: 18), with reference to the Genbank sequence of accession AY274119.3.

  2.3) cDNA corresponding to ORF3, ORF4, ORF7 to ORF11 The same amplification, cloning and sequencing strategy was used to obtain the cDNA fragments respectively corresponding to the following ORFs: ORF3, ORF4, ORF7, ORF8, ORF9 , ORF10 and ORF11. The pairs of primers used for the first amplification are: - ORF3 and ORF4: S / SE / Fl / + / 25069-25088 and S / SE / R1 / - / 26300-26281 - ORF7 to ORF11: S / MN / Fl / + / 26898-26917 and S / MN / R1 / - / 28287-28266 The pairs of primers used for the second amplification are: - ORF3 and ORF4: S / SE / F2 / + / 25110-25129 and S / SE / ORF7 to ORF11: S / MN / F2 / + / 26977-26996 and S / MN / R2 / - / 28218-28199 The conditions of the first amplification (RT-PCR) are as follows: 45 min at 42 ° C., 10 min at 55 ° C., 2 min at 94 ° C. followed by 40 cycles, including a denaturation step at 94 ° C. for 15 sec, a hybridization step at 58 ° C. for 30 sec and an elongation step at 68 ° C. for 1 min, with 5 sec of increment per cycle and finally a step of terminal elongation at 68 ° C. for 7 min. The conditions of the nested PCR are as follows: a denaturation step at 94 ° C. for 2 min was followed by 40 cycles comprising a denaturation step at 94 ° C. for 20 sec, a hybridization step at 58 ° C. for 30 sec and an elongation step at 72 C for 50 sec, with 4 sec of increment per cycle and finally a step of terminal elongation at 72 C for 7 min. The amplification products obtained corresponding to the cDNAs respectively containing the ORF3 and 4 and the ORF7 to 11 were sequenced using the primers: S / SE / + / 25363, S / SE / + / 25835, S / SE / - / 25494, S / SE / - / 25875, S / MN / + / 27839, S / MN / + / 27409, S / MN / - / 27836 S / MN / - / 27799 and cloned as above for other ORFs, to give the plasmids called SARS-SE and SARS-MN. The DNA of these clones was isolated and sequenced using these same primers and universal primers MI3 sense and M13 antisense.

  The sequence of the amplicon representing the cDNA of the region containing the ORFs 3 and 4 (SEQ ID NO: 7) of the strain of SARS-CoV resulting from the sample 031589 has a nucleotide difference with respect to the corresponding sequence of the isolate AY274119-Tor2. This mutation in position 25298 results in a modification of the amino acid sequence of the corresponding protein (ORF 3): in position 11, an arginine (AY274119-Tor2) is changed to glycine in the strain of SARS-CoV resulting from sample n 031589. In contrast, no mutation was identified with respect to the corresponding sequence of isolate AY278741-Urbani. The sequences of the ORFs 3 and 4 the SARS-CoV strain resulting from the sampling No. 031589 respectively correspond to the sequences SEQ ID NO: 10 and 12 in the attached sequence listing.

  The plasmid, called SARS-SE was deposited under No. I-3126, on November 13, 2003, at the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15; it contains the cDNA corresponding to the region between the ORF-5 and the ORF-E and overlapping the ORF-E of the strain of SARS-CoV resulting from the sample indexed under the number 031589, as defined above. above, which region corresponding to the nucleotides of positions 25110 to 26244 (SEQ ID NO: 8), with reference to the Genbank sequence of accession AY274119.3, the amplicon sequence representing the cDNA corresponding to the region containing ORF7 to ORF11 (SEQ ID NO: 19) of the SARS-CoV strain from Collection No. 031589 does not differ from the corresponding sequences of isolates AY274119-Tor2 and AY278741-Urbani. The sequences of the ORF7 to 11 of the strain of SARS-CoV from the sampling No. 031589 correspond respectively to the sequences SEQ ID NO: 22, 24, 26, 28 and 30 in the sequence listing attached.

  The plasmid called SARS-MN was deposited under No. I-3125, on November 13, 2003, with the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15; it contains the cDNA sequence corresponding to the region situated between the ORF-M and the ORF-N of the strain of SARS-CoV resulting from the sample indexed under the number 031589 and taken from Hanoi, as defined above which sequence corresponds to the nucleotides of positions 26977 to 28218 (SEQ ID NO: 20), with reference to the Genbank sequence of accession AY274119.3.

  The sequence of the amplicon representing the cDNA corresponding to the region containing the ORF7 to ORF11 (SEQ ID NO: 19) of the SARS-CoV strain derived from the sample no. 031589 has no differences with respect to the corresponding sequences of the isolates. AY274119-Tor2 and AY278741-Urbani. The sequences of the ORF7 to 11 of the strain of SARS-CoV from the sampling No. 031589 correspond respectively to the sequences SEQ ID NO: 22, 24, 26, 28 and 30 in the sequence listing attached.

  2.4) cDNA Encoding N Protein and Including ORF13 and ORF14 The cDNA was synthesized and amplified as described above for fragments Sa and Sb. More specifically, the reaction mixture containing: 5 l of RNA, 5 μl of H2O ppi 4 μl of 5X reverse transcriptase buffer, 2 μl of dNTP (5 mM), 2 μl of oligo 20T (5 μM) 0.5 l of RNasin (40 IU / ul) and 1.5 l of AMV-RT (10 IU / ul Promega) were incubated in a thermal cycler under the following conditions: 45 min at 42 ° C, 15 min at 55 ° C C, 5 min at 95 C, then it was maintained at +4 C.

  A first PCR amplification was performed with primer pair S / N / F3 / + / 28023 and S / N / R3 / - / 29480.

  The reaction mixture as above for the amplification of the S1 and S2 fragments was incubated in a thermocycler, under the following conditions: an initial denaturation step at 94 ° C. for 2 min was followed by 40 cycles comprising a denaturation step at 94 ° C. for 20 sec, a hybridization step at 55 ° C. for 30 sec and then an extension step at 72 ° C. for 1 min 30 sec with 10 sec of additional elongation at each cycle, followed by a final step of elongation at 72 ° C for 5 min. The amplicon obtained at the first PCR amplification underwent a second PCR amplification step (nested PCR) with primer pairs S / N / F4 / + / 28054 and S / N / R4 / - / 29430 under conditions identical to those of the first amplification.

  The amplification product obtained corresponding to the cDNA coding for the N protein of the strain SARS-CoV resulting from the sample no. 031589 was sequenced using the primers: S / N / F4 / + 128054, S / N / R4 / - / 29430, S / N / + / 28468, S / N / + / 28918 and S / N / - / 28607 and cloned as above for the other ORFs, to give the plasmid called SARS-N. The DNA of these clones was isolated and sequenced using the universal primers M13 sense and M13 antisense, as well as primers S / N / + / 28468, S / N / + 128918 and S / N / - / 28607.

  The sequence of the amplicon representing the cDNA corresponding to the ORF-N and including the ORF13 and ORF14 (SEQ ID NO: 36) of the SARSCoV strain resulting from the sample no. 031589 has no differences with respect to the corresponding sequences. isolates AY274119.3-Tor2 and AY278741-Urbani. The sequence of the N protein of the SARS-CoV strain resulting from the sample no. 031589 corresponds to the sequence SEQ ID NO: 37 in the attached sequence listing.

  The sequences of the ORF13 and 14 of the SARS-CoV strain resulting from the sampling No. 031589 respectively correspond to the sequences SEQ ID NO: 32 and 34 in the attached sequence listing.

  The plasmid called SARS-N was deposited under No. I-3048, on June 5, 2003, with the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15; it contains the cDNA coding for the N protein of the SARS-CoV strain resulting from the sample numbered under 031589, as defined above, which sequence corresponds to the nucleotides of the positions 28054 to 29430 (SEQ ID NO: 38) , with reference to the Genbank sequence of access AY274119.3.

  2.5) Non-coding 5 'and 3' ends a) 5 'non-coding end (5'NC) a1) AD synthesis.

  RNAs from sampling 031589, extracted as above, were subjected to reverse transcription under the following conditions: RNA (15 l) and primer S / L / - / 443 (3 μl at the concentration of 5 ml were incubated for 10 minutes at 75 ° C.

  Next, 5X Reverse Transcriptase Buffer (6 μL, INVITROGEN), 10 mM dNTPs (1 L), 0.1 M DTT (3 L) were added and the mixture was incubated at 50 ° C for 3 min. Finally the reverse transcriptase (3 μl of Superscript, INVITROGEN) was added to the previous mixture which was incubated at 50 ° C. for 1 hour and then at 90 ° C. for 2 minutes. The cDNA thus obtained was purified using the QlAquick PCR purification kit (QIAGEN), according to the manufacturer's recommendations.

  III) Terminal Transferase (TdT) Reaction The cDNA (10 l) is incubated for 2 min at 100 ° C, stored in ice, and then added: H 2 O (2.5 μl), TdT buffer 5 × (4 L, AMERSHAM ), 5mM dATP (2 l) and TdT (1.5 l, AMERSHAM). The mixture thus obtained is incubated for 45 minutes at 37 ° C. and then for 2 minutes at 65 ° C.

  The product obtained is amplified by a first PCR reaction using the primers: S / L / - / 225-206 and anchor 14T: 5'-AGATGAATTCGGTACCTTTTTTTTTTTTTT-3 '(SEQ ID NO: 68). The conditions of the amplification are as follows: an initial denaturation step at 94 ° C. for 2 min is followed by 10 cycles comprising a denaturation step at 94 ° C. for 10 sec, a hybridization step at 45 ° C. for 30 sec then an elongation step at 72 ° C. for 30 sec and then for 30 cycles, comprising a denaturation step at 94 ° C. for 10 sec, a hybridization step at 50 ° C. for 30 sec and then a step of elongation at 72 ° C. for 30 sec, then a final step of elongation at 72 C for 5 min. The product of the first PCR amplification underwent a second amplification step using the primers: S / L / - / 204-185 and 14T anchor mentioned above under conditions identical to those of the first amplification. The amplicon thus obtained was purified, sequenced with the primer S / L / - / 182-163 and then cloned as above for the different ORFs, to give the plasmid called SARS-5 ' NC. The DNA of this clone was isolated and sequenced using the universal primers M13 sense and M13 antisense and the primer S / L / - / l 82-163 supra.

  The amplicon representing the cDNA corresponding to the 5'NC end of the strain of SARS-CoV resulting from the sample numbered under the number 031589 corresponds to the sequence SEQ ID NO: 72 in the attached sequence listing; this sequence does not differ from the corresponding sequences of isolates AY274119.3-Tor2 and AY278741-Urbani.

  The plasmid called SARS-5'NC was deposited under No. I-3124, on November 7, 2003, with the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15; it contains the cDNA corresponding to the 5 'non-coding end of the genome of the SARSCoV strain resulting from the sample indexed under the number 031589, as defined above, which sequence corresponding to the nucleotides of the positions 1 to 204 (SEQ ID NO: 39), referring to the Genbank sequence of access AY274119.3.

  b) 3'non-coding end (3'NC) a1) synthesis of the cDNA The RNAs from the sample 031589, extracted as above, were subjected to reverse transcription, according to the following protocol: the reaction mixture containing RNA (5 μl), H 2 O (5 μl), 5X reverse transcriptase buffer (4 μl), 5 mM dNTP (2 μl), Oligo 20T 5 μM (2 μl), RNasin 40 U / μl (0.5 pl) and RT-AMV 10 IU / l (1.5 l, PROMEGA) was incubated in a thermal cycler under the following conditions: 45 min at 42 C, 15 min at 55 C, 5 min at 95 C, then has been maintained at +4 C.

  The cDNA obtained was amplified by a first PCR reaction using primers S / N / + / 28468-28487 and anchor 14T above. The amplification conditions are as follows: an initial denaturation step at 94 ° C. for 2 minutes is followed by 10 cycles comprising a denaturation step at 94 ° C. for 20 sec, a hybridization step at 45 ° C. for 30 min. then an elongation step at 72 ° C. for 50 sec and then 30 cycles comprising a denaturation step at 94 ° C. for 20 sec, a hybridization step at 50 ° C. for 30 sec and then a step of elongation at 72 ° C. for 50 sec, then a final step of elongation at 72 C for 5 min. The product of the first PCR amplification underwent a second amplification step using primers SIN / + / 28933-28952 and anchor 14T above, under conditions identical to those of the first amplification. The amplicon thus obtained was purified, sequenced with the primer S / N / + / 29257-29278 and cloned as above for the various ORFs, to give the plasmid called SARS-3'NC. The DNA of this clone was isolated and sequenced using the universal primers M13 sense and M13 antisense and primer S / N / + / 29257-29278 supra.

  The amplicon representing the cDNA corresponding to the 3'NC end of the strain of SARS-CoV resulting from the sample numbered under 031589 corresponds to the sequence SEQ ID NO: 73 in the attached sequence listing; this sequence does not differ from the corresponding sequences of isolates AY274119.3-Tor2 and AY278741-Urbani.

  The plasmid called SARS-3'NC was deposited under the number I-3123 on November 7, 2003, from the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15.; it contains the cDNA sequence corresponding to the 3 'non-coding end of the genome of the strain of SARS-CoV resulting from the sample indexed under 031589, as defined above, which sequence corresponds to that located between the nucleotide in position 28933 to 29727 (SEQ ID NO: 40), referring to Genbank sequence access AY274119.3, ends with a series of nucleotides a.

  2.6) ORFla and ORFlb The amplification of the 5 'region containing the ORF1a and ORF1b of the SARS-CoV genome resulting from the sampling 031589 was carried out by performing RT-PCR reactions followed by PCRs nested according to the same principles as those previously mentioned. described for other ORFs. The amplified fragments are overlapping on several tens of bases, thus allowing the computer reconstruction of the complete sequence of this part of the genome. On average, the amplified fragments are two kilobases.

  14 overlapping fragments designated LO to L12 were thus amplified using the following primers: Table II: Primers used for the amplification of the 5 'region (ORF1a and ORF1b) REGION Primer sense Primer antisense Primer sense Primer

AMPLIFIEE

  RT-PCR and RT-PCR nested PCR nonsense PCR SEQUENCEE nested (not including primers) LO SILO / F1 / + 30 SILO / R11-481 50-480 LI S / L1 / F11 + 147 S / L1 / R1 / -2336 S / L11F21 + 211 S / L1 / R21-2241 231-2240 L2 S / L2 / F11 + 2033 S / L21R11-4192 S / L2 / F2 / + 2136 SIL2IR21-4168 2156-4167 L3 S / L3bis / F1 / + 3850 S / L3bis / R1 / -5365 SIL3bis1F21 + 3892 SIL3bisIR2 / -5325 3913-5324 L4b S / L4b / F1 / + 4878 SIL4b / R1 / -6061 S / L4b / F21 + 4932 S / L4b / R21-6024 4952-6023 L4 S / L4 / F1 / + 5272 S / L4 / R11-7392 SIL4 / F21 + 5305 SIL4 / R2 / -7323 5325- 7318 L5 S / L5 / F1 / + 7111 SIL5 / R11-9253 SIL5IF21 + 7275 SIL5 / R21-9157 7296-9156 L6 SIL6 / F11 + 8975 SIL6 / R1 / -11151 SIL6 / F21 + 9032 SIL6IR2 / -11067 9053-11066 L7 S / L71F1 / + 10883 S / L7 / R1 / -13050 S / L7 / F21 + 10928 S / L7 / R2 / -12963 10928-12962 L8 S / L8 / F1 / + 12690 SIL8 / R1I-14857 S / L8 / F2 / + 12815 S / L8 / R2 / - 14835 12835-14834 L9 S / L9 / F11 + 14688 S / L9 / R11-16678 SIL9IF2 / + 14745 SIL9 / R2 / - 16625 14765-16624 LI0 S / L101F11 + 16451 S / L10 / R11-18594 S / L10 / F21 + 16514 SIL10 / R2 / 18571 16534-18570 LI1 S / L11 / F1 / + 18441 S / L11 / R1 / -20612 S / L11 / F2 / + 18500 S / L11 / R2 / - 2 0583 18521-20582 L12 S / L12 / F1 / + 20279 S / L12 / R1 / -22229 S / L12 / F2 / + 20319 S / L12 / R2 / - 22206 20338-22205.

  All fragments were amplified under the following conditions except the LO fragment which was amplified as described above for ORF-M: RT-PCR: 30 min at 42 C, 15 min at 55 C, 2 min at 94 ° C., then the cDNA obtained is amplified under the following conditions: 40 cycles comprising: a denaturation step at 94 ° C. for 15 sec, a hybridization step at 58 ° C. for 30 sec and then an extension step at 68 ° C. C for 1 min 30 sec, with a further 5 sec of elongation at each cycle, then a final step of elongation at 68 C for 7 min. Nested PCR: an initial denaturation step at 94 ° C. for 2 min is followed by 35 cycles comprising: a denaturation step at 94 ° C. for 15 sec, a hybridization step at 60 ° C. for 30 sec and then a step of elongation at 72 C for 1 min 30 sec, with 5 sec further elongation at each cycle, then a final elongation step at 72 C for 7 min. The amplification products were sequenced using the primers defined in Table III below: Table III: Primers used for sequencing the 5 'region (ORF1a and ORF1b) 10 Sequence Names (SEQ ID NO: 76 to 139) S / L31 + 14932 5'-CCACACACAGCTTGTGGATA-3 'SlL4 / + 16401 5'-CCGAAGTTGTAGGCAATGTC-3' SIL4 / + 16964 5'-TTTGGTGCTCCTTCTTATTG-3 'SIL4 / - / 6817 5'-CCGGCATCCAAACATAATTT-3' / L5 / - 17633 5'-TGGTCAGTAGGGTTGATTGG-3 'S / L5 / -18127 5'-CATCCTTTGTGTCAACATCG-3' S / L5 / -18633 5'-GTCACGAGTGACACCATCCT-3 'S / L5 / + / 7839 5'ATGCGACGAGTCTGCTTCTA-3 S / L5 / + 18785 5'-TTCATAGTGCCTGGCTTACC-3 'S / L5 / + 18255 5'-ATCTTGGCGCATGTATTGAC-3' S1L6 / - / 9422 5'-TGCATTAGCAGCAACAACAT-3 'S / L6 / - / 9966 5'-TCTGCAGAACAGCAGAAGTG 5'-CCTTGTGGCAATGAAGTACA-3 'S / L6 / + / 10105 5'-ATGTCATTTGCACAGCAGAA-3' S / L6 / + / 9571 -CTTCAATGGTTTG CCATGTT-3 'S / L7 / -111271 5'-TGCGAGCTGTCATGAGAATA-3' SIL7 / -111801 5'AACCGAGAGCAGTACCACAG-3 'SIL71- / 12383 5'-TTTGGCTGCTGTAGTCAATG-3' S / L7 / + 112640 5'-CTACGACAGATGTCCTGTGC-3 'SIL7 / + / 12088 5'-GAGCAGGCTGTAGCTAATGG-3' S / L7 / + / 11551 5'-TTAGGCTATTGTTGCTGCTG-3 'S / L8 / -13160 5'CAGACAACATGAAGCACCAC-3' S / L81- 5'-CGCTGACGTGATATATGTGG-3 'S / L8 / 14284 5'-TGCACAATGAAGGATACACC-3' SIL8 / + / 14453 5'-ACATAGCTCGCGTCTCAGTT-3 'S / L8 / + 113968 5'-GGCATTGTAGGCGTACTGAC-3' S / L8 / + 113401 5'GTTTGCGGTGTAAGTGCAG-3 'S / L9 / -15098 5'-TAGTGGCGGCTATTGACTTC-3' S / L9 / - 15677 5'-CTAAACCTTGAGCCGCATAG-3 'S / L9 / -16247 5'-CATGGTCATAGCAGCACTTG-3' S / L9 / +16323 5'-CCAGGTTGTGATGTCACTGAT-3 'S / L9 / + 15858 5'CCTTACCCAGATCCATCAAG-3' S / L9 / + 15288 5'-CGCAAACATAACACTTGCTG-3 'S / L10 / 16914 5'-AGTGTTGGGTACAAGCCAGT-3' S / L10 / -17466 5'-GTTCCAAGGAACATGTCTGG-3 'S / L10 / -18022 5'-AGGTGCCTGTGTAGGATGAA-3' S / L10 / + 18245 5'GGGCTGTCATGCAACTAGAG-3 'S / L10 / + 17663 5'-TCTTACACGCAATCCTGCTT-3' 2862981 44 sequences of the LO to L12 fragments of the strain of SARS-CoV resulting from the sample indexed under the number 031589, respectively correspond to the sequences SEQ ID NO: 41 to SEQ ID NO: 54 in the list of e attached sequence. Among these sequences, only that corresponding to the L5 fragments has a nucleotide difference with respect to the corresponding sequence of the AY278741-Urbani isolate. This tic mutation at position 7919 results in a modification of the amino acid sequence of the corresponding protein, encoded by ORF 1a: at position 2552, a valine (codon gtt; AY278741) is changed to alanine (codon gct) in the strain SARS-CoV 031589. In contrast, no mutation was identified with respect to the corresponding sequence of isolate AY274119. 3-Urbani. The other fragments do not differ from the corresponding sequences of the Tor2 and Urbani isolates.

  SIL10 / + 17061 S / L1 1 / -11 8877 S / L 1 1 / -19396 S / L11 / -20002 S / L11 / + 20245 SIL111 + 119611 S / L11 / + / 19021 SARS / L1 / F3 / + 800 SARS / L1 / F41 + 1391 SARS / L11F51 + 1925 SARS / L1 / R3 / -1674 SARS / L1 / R41-1107 SARS / L1 / R5 / -520 SARS / L21F3 / + 2664 SARS / L2 / F4 / + 3232 SARS / L2 / F5 / + 3746 SARS / L2 / R3 / -3579 SARSIL2 / R4 / -2991 SARS / L2 / R5 / -2529 SARS / L3 / F3 / + 4708 SARS / L3 / F4 / + 5305 SARS / L3 / F5 / + 5822 SARS / L 3 / R3 / -561 0 SARS / L3 / R4 / -4988 SARS / L3 / R5 / -4437 5'-TACCCATCTG CTCG CATAGT-3 '5'-GCAAG CAGAATTAACCCTCA-3' 5'-AG CACCACCTAAATTG CATC-3 '5'-TGGTCCCTTTGAAGGTGTTA-3' 5'-TCGAACACAT CGTTTATG GA-3 '5'-GAAG CACCT GTTTCCATCAT-3' 5'-ACGATG CTCAG CCATGTAGT-3 '5'GAGGTGCAGTCACTCGCTAT-3' 5'-CAGAGATTGGACCTGAGCAT- 3 '5'CAGCAAACCACTCAATTCCT-3' 5'-AAATGATG GCAACCTCTT CA-3 '5'CACGTGGTTGAATGACTTTG-3' 5'-ATTTCTG CAACCAGCT CAAC-3 '5'CGCATTGTCTCCTGGTTTAC-3' 5'-GAGATTGAGCCAGAACCAGA-3 '5'ATGAGCAGGTTGTCATGGAT-3 CTCTAGGAGTAGGAGGG ATG-3 '5'TTTCTTCACCAGCATCATCA-3' 5'-CACCGTTCTTGAGAACAACC-3 '5'TCTTTGGCTGGCTCTTACAG-3' 5'-GCTGGTGATGCTGCTAACTT-3 '5' CCATCAAGCCTGTGTCGTAT-3 '5'-CAGGTGGTGCAGACATCATA-3' 5'AACATCAGCACCATCCAAGT-3 '5'-ATCGGACACCATAGTCAACG-3' 2862981 45 Example 2: Production and Purification of Recombinant N and S Proteins of the SARS-CoV Strain Derived from the Sample Listed Under No. 031589 The whole protein and two polypeptide fragments of the S protein of the SARS-CoV strain from the sample numbered 031589 were produced in E. coli, in the form of fusion proteins comprising a polyhistidine N-label or C-terminus. In both S polypeptides, the N- and C-terminal hydrophobic sequences of the S protein (signal peptide: positions 1 to 13 and transmembrane helix: positions 1196 to 1218) were deleted whereas the p helix (positions 565 to 687) was deleted. and the two coiled-coil units (positions 895 to 980 and 1155 to 1186) of the S protein have been preserved. These two polypeptides are constituted by: a long fragment (SL) corresponding to positions 14 to 1193 of the amino acid sequence of protein S and a short fragment (Sc) corresponding to positions 475 to 1193 of the amino acid sequence of Protein S. 1) Cloning of the N, SL and Sc cDNAs into the expression vectors pIVEX2.3 and pIVEX2.4. The cDNAs corresponding to the N protein and to the SL and Sc fragments were amplified by PCR under standard conditions. using Platinium Pfx DNA polymerase (INVITROGEN). Plasmids SARS-N and SARS-S were used as template and the following oligonucleotides as primers: 5'-CCCATATGTCTGATAATGGACCCCAATCAAAC-3 '(N sense, SEQ ID NO: 55) 5'CCCCCGGGTGCCTGAGTTGAATCAGCAGAAGC-3' (N antisense, SEQ ID NO: 56) 5'CCCATATGAGTGACCTTGACCGGTGCACCAC-3 '(Sc sense, SEQ ID NO: 57) 5'CCCATATGAAACCTTGCACCCCACCTGCTC-3' (SL sense, SEQ ID NO: 58) 5'CCCCCGGGTTTAATATATTGCTCATATTTTCCC-3 '(Sc and SL antisense, SEQ ID NO: 59).

  The sense primers introduce an Ndel (underlined) site while the antisense primers introduce an XmaI or SmaI (underlined) site. The 3 amplification products were purified on a column (QlAquick PCR Purification kit, QIAGEN) and cloned into an appropriate vector. The purified plasmid DNA of the 3 constructs (QIAFilter Midi Plasmid kit, QIAGEN) was verified by sequencing and digested with the NdeI and XmaI enzymes. The 3 fragments corresponding to the N, SL and Sc cDNAs were purified on an agarose gel and then inserted into the plasmids pIVEX2.3MCS (C-terminal polyhistidine label) and pIVEX2.4d (N-terminal polyhistidine label) previously digested with the same enzymes. After verifying the constructions, the 6 expression vectors thus obtained (pIV2.3N, pIV2.3Sc, pIV2.3SL, pIV2.4N, pIV2.4Sc also called pIV2.4S1, pIV2.4SL) were then used, from one part to test the expression of the proteins in-vitro, and secondly to transform the bacterial strain BL21 (DE3) pDIA17 (NOVAGEN). These constructs encode proteins whose expected molecular weight is: pIV2.3N (47174 Da), pIV2.3Sc (82897 Da), pIV2. 3SL (132056 Da), pIV2.4N (48996 Da), pIV2.4S1 (81076 Da) and pIV2. 4SL (133877 Da).

  2) Analysis of the expression of recombinant proteins in vitro and in vivo The expression of recombinant proteins from the 6 recombinant vectors was first tested in an invitro system (RTS100, Roche). The proteins produced in vitro, after incubation of the pIVEX recombinant vectors, 4 h at 30 ° C., in the RTS 100 system, were analyzed by Western blot using an anti-(his) 6 antibody coupled to peroxidase. . The in vitro expression result (FIG. 1) shows that only the N protein is expressed in large quantities, regardless of the position, N- or C-terminal, of the polyhistidine label. In a second step, the expression of the N and S proteins was tested in vivo at 30 ° C. in LB medium, in the presence or absence of inducer (1 mM IPTG). N protein is very well produced in this bacterial system (Figure 2) and is found mainly in a soluble fraction after lysis of bacteria. On the other hand, the long version of S (SL) is very little produced and completely insoluble (Figure 3). The short version (Sc) also has a very low solubility, but a much higher expression rate than the long version. Moreover, the Sc construct fused to a polyhistidine label in the C-terminal position is smaller than expected. An immunodetection experiment with an anti-polyhistidine antibody showed that this construct was incomplete. In conclusion, the two constructs, pIV2.3N and pIV2.4S1, expressing respectively the entire N protein fused to the C-terminal polyhistidine tag and the short S protein fused to the N-terminal polyhistidine tag, were retained. to produce both proteins in large quantities to purify them.

  3) Analysis of the Antigenic Activity of the Recombinant Proteins The antigenic activity of the N, SL and Sc proteins was tested by Western blot, using two serum samples, from the same patient infected with SARS-CoV, taken 8 days (M12) and 29 days- (M13) after the onset of SARS symptoms. The experimental protocol is as described in Example 3. The results illustrated in FIG. 4 show (i) seroconversion of the patient, and (ii) that the N protein has a higher antigenic reactivity than the short S protein.

  4) Purification of the N Protein from pIV2.3N Several N protein purification experiments, produced from the pIV2.3N vector, were carried out according to the following protocol. BL21 (DE3) pDIA17 bacteria, transformed with the pIV2 expression vector. 3N, were grown at 30 C in 1 liter of culture medium containing 0.1 mg / ml ampicillin, and induced by 1 mM IPTG when the cell density, equivalent to A600 = 0.8, is reached (about 3 hours). After 2 hours of culture in the presence of inducer, the cells were recovered by centrifugation (10 min at 5000 rpm), resuspended in the lysis buffer (50 mM NaH2PO4, 0.3 M NaCl, 20 mM imidazole, pH 8 containing the mixture of protease inhibitors Complete, Roche), and lysed by the French press (12000 psi). After centrifugation of the bacterial lysate (15 min at 12000 rpm), the supernatant (50 ml) was deposited at a flow rate of 1 ml / min on a column (15 ml) of metal chelation (Ni-NTA superflow, Qiagen), equilibrated with the lysis buffer. After washing the column with 200 ml of lysis buffer, the N protein was eluted with an imidazole gradient (20 x 250 mM) in 10 column volumes. The fractions containing the N protein were pooled and analyzed by polyacrylamide gel electrophoresis under denaturing conditions followed by staining with Coomassie blue. The results illustrated in FIG. 5 show that the protocol used makes it possible to purify the N protein with a very satisfactory homogeneity (95%) and an average yield of 15 mg of protein per liter of culture.

  5) Purification of the Sc protein from pIV2.4Sc (pIV2.4S1) The protocol followed to purify the short S protein is very different from that described above because the protein is strongly aggregated in the bacterial system (body inclusion). The BL21 (DE3) pDIA17 bacteria, transformed with the pIV2.4S1 expression vector, were cultured at 30 ° C. in 1 liter of culture medium containing 0.1 mg / ml ampicillin and induced by 1 mM IPTG. when the cell density, equivalent to A600 = 0.8, is reached (approximately 3 hours). After 2 hours of culture in the presence of inducer, the cells were recovered by centrifugation (10 min at 5000 rpm), resuspended in the lysis buffer (0.1 M Tris-HCl, 1 mM EDTA, pH 7, 5), and lysed by French press (1200 psi). After centrifugation of the bacterial lysate (15 min at 12000 rpm), the pellet was resuspended in 25 ml of lysis buffer containing 2% Triton X100 and 10 mM [3-mercaptoethanol, followed by centrifugation for 20 min at 12000 rpm. The pellet was resuspended in 10 mM Tris-HCl buffer containing 7 M urea, and gently stirred for 30 min at room temperature. This last wash of inclusion bodies with 7 M urea is necessary to remove most membrane proteins from E. coli which co-sediment with the aggregated protein Sc. After a final centrifugation for 20 min at 12000 rpm, the final pellet is resuspended in 10 mM Tris-HCl buffer. The electrophoretic analysis of this preparation (FIG. 6) shows that the short S protein can be purified with a satisfactory homogeneity (approximately 90%) from the inclusion bodies (insoluble extract).

  Example 3 Immunodominance of the N Protein The reactivity of the antibodies present in the serum of patients suffering from atypical pneumonitis caused by the SARS-associated coronavirus (SARSCoV), with respect to the various proteins of this virus, was analyzed by Western -blot under the conditions described below.

  1) Material a) lysate of cells infected with SARS-CoV Vero E6 cells (2x106) were infected with SARS-CoV (isolate listed under the number FFM / MA104) at a multiplicity of infection (MOI) of 10 -1 or 10-2 and then incubated in DMEM medium containing 2% FCS at 35 ° C. in an atmosphere containing 5% CO 2. 48 hours later, the cell layer was washed with PBS and then lysed with 500 μl of Laemmli deposition buffer and containing 13-mercaptoethanol. The samples were then boiled for 10 minutes and then sonicated 3 times 20 seconds.

  b) antibody b1) Patient serum atypical pneumonitis The serum referenced at the National Influenza Reference Center (Region-Nord) under N 20033168 is that of a French patient suffering from atypical pneumonitis caused by SARS-CoV taken at day 38 after the onset of symptoms; the diagnosis of SARS-CoV infection was made by nested RT-PCR and quantitative PCR.

  b2) Polymeric sera of the mouse-protein source directed to the proteinine and the S-proteinine The sera are those produced from the recombinant proteins N and Sc (Example 2), according to the immunization protocol described in Example 4; this is rabbit serum P13097 (anti-N serum) and rabbit serum P11135 (anti-S serum). 2) Method 1 of lysate of cells infected with SARS-CoV at M.O.I. 10.1 and 10-2 and, as a control, 20 l of a lysate of uninfected cells (mock) were separated on a SDS gel with 10% polyacrylamide and then transferred onto a nitrocellulose membrane. After blocking in a solution of PBS / milk 5% / 0.1% Tween and washing in PBS / 0.1% Tween, this membrane was hybridized overnight at 4 ° C. with: (i) the immunoserum N 20033168 diluted 1/300, 1/1000 and 1/3000 in PBS / BSA buffer 1% / Tween 0.1%, (ii) serum of rabbit P13097 (anti-N serum) diluted to 1/50000 in the same buffer and (iii) rabbit serum P11135 (anti-S serum) diluted 1/10000 in the same buffer. After washing in PBS / Tween, secondary hybridization was carried out using either polyclonal sheep antibodies directed against the heavy and light chains of human immunoglobulin G and coupled to peroxidase (NA933V, Amersham), or polyclonal donkey antibodies to the heavy and light chains of rabbit immunoglobulin G coupled to peroxidase (NA934V, Amersham). The fixed antibodies were revealed using the ECL + kit (Amersham) and Hyperfilm MP autoradiography films (Amersham). A molecular weight scale (kDa) is shown in the figure.

  3) Results Figure 7 shows that three 35, 55 and 200 kDa apparent molecular weight polypeptides are specifically detected in extracts of SARS-CoV infected cells.

  In order to identify these polypeptides, two other immunoblots (FIG. 8) were made on the same samples and under the same conditions with polyclonal rabbit antibodies specific for nucleoprotein N (rabbit P 13097, FIG. 8A) and the spike S (rabbit P11135, FIG. 8B) This experiment shows that the 200 kDa polypeptide corresponds to the spicule glycoprotein S of SARS-CoV, that the 55 kDa polypeptide corresponds to nucleoprotein N while the 35 kDa polypeptide probably represents A truncated or degraded form of N. The data presented in Figure 7 thus show that serum 20033168 reacts strongly with N and much more weakly with S of SARS-CoV, since the 35 and 55 kDa polypeptides are revealed under the form of intense bands for 1/300, 1/1000 and 1/3000 dilutions of the antiserum whereas the 200 kDa polypeptide is only weakly revealed for a diluate 1/300. It may also be noted that no other SARS-CoV polypeptide is detected for dilutions greater than 1/300 of the 20033168 serum.

  This experiment indicates that the SARS-CoV N-specific antibody response dominates the antibody responses specific to other SARS-CoV polypeptides and in particular the antibody response to the S-glycoprotein. It indicates immunodominance of the N-nucleoprotein human infections with SARS-CoV.

  Example 4 Preparation of Monospecific Polyclonal Antibodies Directed Against SARS-Associated Coronavirus-Associated N and S Proteins (SARS-CoV) 1) Material and Method Three rabbits (P13097, P13081, P13031) were immunized with the purified recombinant polypeptide corresponding to the entire nucleoprotein (N), prepared according to the protocol described in Example 2. After a first injection of 0.35 mg per rabbit of protein emulsified in complete Freund's adjuvant (intradermal route), the animals were received 3 booster injections at 3 and 4 weeks apart, 0.35 mg of recombinant protein emulsified as incomplete Freund's adjuvant.

  Three rabbits (P11135, P13042, P14001) were immunized with the recombinant polypeptide corresponding to the short fragment of the protein S (Sc), produced as described in Example 2. As this polypeptide is found mainly in the form of body of inclusion in the bacterial cytoplasm, the animals received 4 intra-dermal injections at 3-4 week intervals of an inclusion body preparation corresponding to 0.5 mg of recombinant protein emulsified as incomplete Freund's adjuvant. The first 3 injections were carried out with an inclusion body preparation prepared according to the protocol described in Example 2, while the fourth injection was carried out with an inclusion body preparation which were prepared according to the described protocol. in Example 2 and then purified on a sucrose gradient and washed in 2% Triton X100.

  For each rabbit, a pre-immune serum (p.i.) was prepared prior to the first immunization and an immun-serum (I.S.) 5 weeks after the fourth immunization.

  In a first step, the reactivity of the sera was analyzed by ELISA test with respect to recombinant protein preparations similar to those used for the immunizations; the ELISA tests were carried out according to the protocol and with the reagents as described in Example 6.

  In a second step, the reactivity of the sera was analyzed by performing an immunoblot (western blot) of a lysate of cells infected with SARS-CoV, following the protocol as described in Example 3.

  2) Results The ELISA tests (FIG. 9) demonstrate that the recombinant protein N and inclusion body preparations of the short fragment of the S (Sc) protein are immunogenic in the animal and that the titre of the immune sera is high ( more than 1/25000).

  The immunoblotting (FIG. 8) shows that the rabbit immune serum P13097 recognizes two polypeptides present in the lysates of cells infected with SARS-CoV: a polypeptide whose apparent molecular mass (50-55 kDa according to the experiments) is compatible with that of nucleoprotein N (422 residues, predicted molecular mass of 46 kDa) and a polypeptide of 35 kDa, which presumably represents a truncated or degraded form of N. This experiment also shows that the serum of rabbit P11135 mainly recognizes a polypeptide whose apparent molecular mass (180-220 kDa according to the experiments) is compatible with a glycosylated form of S (1255 residues, unglycosylated polypeptide chain of 139 kDa), as well as lighter polypeptides, which presumably represent truncated forms and / or non-glycosylated S. In conclusion, all of these experiments demonstrate that recombinant polypeptides and Xpressed in E. coli and corresponding to the N and S SARS-CoV proteins can induce in the animal polyclonal antibodies capable of recognizing the native forms of these proteins.

  Example 5 Preparation of Monospecific Polyclonal Antibodies Directed Against SARS-Associated Coronavirus (SARS-CoV) M and E Proteins 1) Analysis of M and E Protein Structure a) Protein E Structure of SARS-Protein E CoV (76 amino acids) was analyzed in silico, using various software such as SignalP v1.1, NetNGlyc 1.0, THMM 1.0 and 2.0 (Krogh et al., 2001, J. Mol Biol., 305 (3)). ): 567-580) or TOPPRED (von Heijne, 1992, J. Mol Biol 225, 487-494). The analysis shows that this non-glycosylated polypeptide is a type 1 membrane protein, containing a single transmembrane helix (aa 12-34 according to THMM), and the major part of the hydrophilic domain (42 residues) is localized to the membrane. C-terminus and presumably within the viral particle (endodomain). We can notice an inversion in the topology predicted by the versions 1.0 (N-ter is external) and 2.0 (N-ter is internal) of the THMM software, but that other algorithms, in particular TOPPRED and THUMBUP (Zhou and Zhou , 2003, Protein Science 12: 1547-1555) confirm an external location of the N-terminus of E. b) Protein M A similar analysis carried out on the SARS-CoV M protein (221 amino acids) shows that this polypeptide does not have a signal peptide (according to the signalP vl.l software) but three transmembrane domains (residues 15-37, 50-72, 2862981 53 77-99 according to THMM2.0) and a large hydrophilic domain (aa 100-221) located inside the viral particle (endodomain). It is presumably glycosylated on asparagine at position 4 (according to NetNGlyc 1.0).

Thus, in agreement with the experimental data known for the other coronaviruses, it is remarkable that the two M and E proteins have endodomains corresponding to most polypeptides and ectodomains of very small size.

  the ectodomain of E probably corresponds to residues 1 to 11 or 1 to 12 of the protein: MYSFVSEETGT (L), SEQ ID NO: 70. Indeed, the probability associated with the transmembrane localization of residue 12 is intermediate (0 , 56 from THMM 2.0).

  the ectodomain of M probably corresponds to residues 2 to 14 of the protein: ADNGTITVEELKQ, SEQ ID NO: 69. Indeed, the N-terminal methionine of M is very probably cleaved from the mature polypeptide because the residue in position 2 is an alanine ( Varshavsky, 1996, 93: 12142-12149).

  Moreover, the analysis of the hydrophobicity (Kyte & Doolittle, Hopp & Woods) of the protein E shows that the C-terminal end of the endodomain of E is hydrophilic and therefore presumably exposed on the surface of this protein. field. Thus, a synthetic peptide corresponding to this end is a good immunogenic candidate for inducing antibodies directed against the endodomain of E. As a result, a peptide corresponding to the 24 C-terminal residues of E was synthesized.

  2) Preparation of Antibodies Against the Ectodomain of the M and E Proteins and the Endodomain of the E Protein M2-14 Peptides (GITVEELKQ DNA, SEQ ID NO: 69), EI-12 (MYSFVSEETGTL, SEQ ID NO: 70 ) and E53-76 (KPTVYVYSRV KNLNSSEGVP DLLV, SEQ ID NO: 71) were synthesized by Neosystem. They were coupled to KLH (Keyhole Limpet Hemocyanin) using MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester) via a cysteine added during either N-terminal synthesis of the peptide (case of E53-76 ) or in C-terminal (case of M2-14 and El-12).

  Two rabbits were immunized with each of the conjugates, following the following immunization protocol: after a first injection of 0.5 mg peptide coupled to KLH and emulsified in Freund's complete adjuvant (intradermal route), the animals receive 2 to 4 booster injections at 3 or 4 week intervals of 0.25 mg of peptide coupled to KLH and emulsified as incomplete Freund's adjuvant.

  For each rabbit, a pre-immune serum (pi) was prepared prior to the first immunization and an immune serum (I.S.) was prepared 3 to 5 weeks after the booster shots.

  The reactivity of the sera is first analyzed by ELISA with respect to the peptide used for the immunization, then by immunoblotting with lysates of cells infected with SARS-CoV, as described for the antiserum sera. N and anti-S of Example 4, according to similar protocols to those described in Examples 3 and 6, respectively for immunoblotting and ELISA.

  In a second step, the reactivity of antisera directed against peptides M2-14 and El-12 to recognize the ectodomains of M and E present on the surface of the native viral particle is analyzed by immunocapture and / or immunoprecipitation of native virions.

  Example 6: ELISA Reactivity Analysis of Recombinant N-Protein Against Sera from SARS Patients 1) Material The antigen used to prepare the solid phases is the purified recombinant nucleoprotein N prepared according to the protocol described in Example 2.

  The sera to be tested (Table IV) were chosen on the basis of the results of analysis of their immunofluorescence reactivity (titre IF-SARS), vis-à-vis lysates of cells infected with SARS-CoV. 15

  TABLE IV Serum Tested by ELISA Reference N Serum Type Date of Title IF-SARS serum Serum *** 3050 A Control na * nt ** 3048 B Control na nt 033168 D Patient 1- SARS 27/04/03 ( J38) 320 033397 E Patient-1 SARS 11/05/03 (J52) 320 032632 F Patient-2 SARS 21/03/03 (D17) 2500 032791 G Patient-3 SARS 04/04/03 (D3) <40 033258 H Patient-3 SARS 28/04/03 (D27) 160 * na: not applicable. ** nt: not tested. *** the dates shown are the number of days after the onset of SARS symptoms.

  2) Method Protein N (100 μl) diluted at different concentrations in 0.1 M carbonate buffer, pH 9.6 (1, 2 or 4 μg / ml) is distributed in the wells of ELISA plates, then the plates are incubated overnight at laboratory temperature.

  Plates are washed with PBS-Tween buffer, saturated with PBS-skim-sucrose (5%). The sera to be tested (100 l) previously diluted (1/50, 1/100, 1/200, 1/400, 1/800, 1/1600 and 1/3200) are added, then the plates are incubated for 1 hour at C. After 3 washes, the anti-human IgG conjugate labeled with peroxidase (reference 209-035098, JACKSON) diluted 1/18000 is added and the plates are incubated for 1h at 37 C. After 4 washes, the chromogen (TMB ) and the substrate (H2O2) are added and the plates are incubated for 30 min at room temperature, protected from light. The reaction is then stopped and the absorbance at 450 nm is measured using an automatic reader.

  3) Results The ELISA tests (FIG. 10) demonstrate that the recombinant protein N preparation is recognized specifically by the antibodies of sera of SARS patients taken in the late phase of the infection (> 17 days after the onset of symptoms). that it is not significantly recognized by the antibodies of a patient serum taken early in the infection (3 days after the onset of symptoms) or by control sera from subjects without SARS.

  Example 7: Detection of SARS-CoV Associated Coronavirus (SARS-CoV) by Real-Time RTPCR Using Primers Specific to the Nucleoprotein Gene 1) Development of RT-PCR Conditions a) Primer Design and probes The design of the primers and probes was carried out from the genome sequence of the strain of SARS-CoV resulting from the sampling indexed under the number 031589, using the program "Light Cycler Probe Design (Roche)". Thus the following two sets of primers and probes were selected: - series 1 (SEQ ID NO: 60, 61, 64, 65): sense primer: N / + / 28507: 5'-GGC ATC GTA TGG GTT G -3 '[28507-28522] - antisense primer: NI- / 28774: 5'-CAG TTT CAC CAC CTC C-3' [28774-28759] probe 1: 5'-GGC ACC CGC AAT CCT AAT AAC AAT GC- fluorescein 3 '[28561-28586] probe 2: 5' Red705 -GCC ACC GTG CTA CAA CTT CCT-phosphate [28588-28608] series 2 (SEQ ID NO: 62, 63, 66, 67) - sense primer: N / + / 28375: 5'-GGC TAC TAC CGA AGA G-3 '[28375-28390] - antisense primer: NI- / 28702: 5'-AAT TAC GAC GAC TAC G-3' [28702-28687] - probe 1 : SARS / N / FL: 5'-ATA CAC CCA ACC AAG ACCG TTG GC - fluorescein 3 '[28541-28563] - probe 2: SRASINILC705: 5' Red705-CCC GCA ATC CTA ATA ACA ATG CTG C-phosphate 3 ' [28565-28589] b) Analysis of the Effectiveness of the Two Primer Couples In order to test the respective efficacy of the two primer pairs, an amplification by RT-PCR was performed on a synthetic RNA corresponding to the nucleotides 28054-29430 of the genome of the SARS-CoV strain from the sample numbered 031589and containing the sequence of the N gene. More precisely: This synthetic RNA was prepared by in vitro transcription with the aid of the T7 phage RNA polymerase, a DNA template obtained by linearization of the plasmid SARS-N with the enzyme Bam HI. After removal of the DNA template by digestion with DNAse 1, the synthetic RNAs are purified by phenol-chloroform extraction followed by two successive precipitations of ammonium acetate and isopropanol. They are then quantified by absorbance measurement at 57 nm and their quality is controlled by the absorbance ratio at 260 and 280 nm as well as by agarose gel electrophoresis. Thus, the concentration of the synthetic RNA preparation used for these studies is 1.6 mg / ml, which corresponds to 2.1.1 015 copies / ml of RNA.

  Decreasing amounts of synthetic RNA were amplified by RTPCR using the "SuperscriptTM One-Step RT-PCR with Platinum Taq" kit and primer pairs # 1 (N / + / 28507, N / - / 28774). ) (FIG. 1A) and No. 2 (NI + / 28375, N / - / 28702) (FIG. 1B), following the indications of the supplier. The amplification conditions used are as follows: the cDNA was synthesized by incubation for 30 minutes at 45 ° C., 15 minutes at 55 ° C. and then for 2 minutes at 94 ° C. and then amplified by 5 cycles comprising: a denaturation step at 94 C for 15 sec, a hybridization step at 45 C for 30 sec and then an extension step at 72 C for 30 sec, followed by 35 cycles comprising: a denaturation step at 94 C for 15 sec, a step of hybridization at 55 ° C. for 30 sec then an extension step at 72 ° C. for 30 sec, with 2 sec of additional elongation at each cycle, and a final elongation step at 72 ° C. for 5 min. The amplification products obtained were then maintained at 10 ° C.

  The results presented in FIG. 11 show that pair of primers n 2 (NI + / 28375, N / - / 28702) can detect up to 10 copies of RNA (low intensity band) or 102 copies (band of good intensity) against 104 copies for primer pair # 1 (N / + / 28507, N / - / 28774). The amplicons are respectively 268 bp (torque 1) and 328 bp (torque 2).

  c) development of real-time RT-PCR Real-time RT-PCR was developed using primer pair n 2 and the probe pair consisting of SARS / N / FL and SARS / N / LC705 (Figure 2).

  The amplification was carried out on a LightCyclerTM (Roche) using the "Light Cycler RNA Amplification Kit Hybridization Probes" kit (reference 2,015,145, Roche) under the following optimized conditions. A reaction mixture containing: H 2 O (6.8 L), 25 mM MgCl 2 (0.8 L, final 4 Mg 2+), 5X reaction mixture (4), 3 M SARS / N / FL probe (0, 5 μl, 0.075 M final), 58 M SARAS / N / LC705 probe (0.5 l, final 0.075 M), 10M N / + / 28375 primer (1 L, final 0.5 M), N primer / - / 28702 10 M (1 l, 0.5 M final), enzyme mixture (0.4 l) and sample (viral RNA, 5 l) was amplified by following the following program: -Inverse transcription: 50 C 10: O0min analysis mode: none - Denaturation: 95 C 30sec xl analysis mode: none - Amplification: 95 C 2sec 50 C 15sec analysis mode: quantification * - x45 72 C 13sec thermal ramp 2.0 C / sec cooling: 40 C 30sec xl analysis mode: none * The fluorescence measurement is done at the end of the hybridization and at each cycle (in SINGLE mode).

  The results presented in FIG. 12 show that this RT-PCR in real time is very sensitive since it makes it possible to detect 102 copies of synthetic RNA in 100% of the analyzed samples (29/29 samples in 8 experiments) and up to 10 copies of RNA in 100% of the 5 samples analyzed (40/45 samples in 8 experiments). It also shows that this RT-PCR makes it possible to detect the presence of the SARS-CoV genome in a sample and to quantify the number of genomes present. By way of example, the viral RNA of a stock of SARSCoV grown on Vero E6 cells was extracted using the "Qiamp viral RNA extraction" kit (Qiagen), diluted to 0.05 × 10 4 and analyzed by RT- Real-time PCR according to the protocol described above; the analysis presented in Figure 12 shows that this virus stock contains 6.5.109 equivalent genomes / ml (geq / ml), which is quite similar to the value of 1.0.1010 geq / ml measured using of the "RealArtTM HPACoronavirus LC RT PCR Reagents" kit marketed by Artus.

  d) detection of SARS-CoV RNA by real-time PCR from respiratory samples A comparative study was carried out on a series of respiratory samples received by the National Influenza Reference Center (North Region) and likely to contain SARS-CoV. To do this, the RNA was extracted from the samples using the "Qiamp viral RNA extraction" kit (Qiagen) and analyzed by RT-PCR in real time, on the one hand using pairs of primers. and probes of the series n 2 under the conditions described above on the one hand, and on the other hand using the kit "LightCycler SARS-CoV quantification kit" marketed by Roche (reference 03 604 438) . The results are summarized in the Table below. They show that 18 of the 26 samples are negative and 5 of the 26 samples are positive for both kits, while one sample is positive for the only Roche kit and two samples for the only "series2" reagents. In addition, for 3 samples (20032701, 20032712, 20032714) the detected amounts of RNA are clearly higher with the reagents (probes and primers) of the n 2 series. These results indicate that the N "series2" primers and probes are more sensitive for the detection of the genome of SARS-CoV in biological samples than those of the kit currently available.

  Table V: Real-time RT-PCR analysis of the RNAs extracted from a series of samples of 5 patients using pairs of primers and probes of the series n 2 (N "series 2") or of the kit "LightCycler SARS-CoV Quantization Kit" (Roche). The type of sample is indicated as well as the number of viral genome copies measured in each of the two tests. NEG: negative RT-PCR.

  Collection n Patient Collection Specimen ROCHE KIT N "series2" 20033082 K nasal NEG NEG 20033083 K pharyngeal NEG NEG 20033086 K nasal NEG NEG 20033087 K pharyngeal NEG NEG 20032802 M nasal NEG NEG 20032803 M expectoration NEG NEG 20032806 M nasal or pharyngeal NEG NEG 20031746ARN2 C pharyngeal NEG NEG 20032711 C nasal or pharyngeal 39 NEG 20032910 B nasal NEG NEG 20032911 B pharyngeal NEG NEG 20033356 Sputum NEG NEG 20033357 V expectoration NEG NEG 20031725 K asp. endotracheal NEG 150 20032657 K asp. endotracheal NEG NEG 20032698 K asp. endotracheal NEG NEG 20032720 K asp. endotracheal 3 5 20033074 K stool 115 257 20032701 M pharyngeal 443 1676 20032702 M expectoration NEG 249 20031747ARN2 C pharyngeal NEG NEG 20032712 C unknown 634 6914 20032714 C pharyngeal 17 223 20032800 B nasal NEG NEG 20033353 N nasal NEG NEG 20033384 N nasal NEG NEG 2862981 60

Claims (5)

  1) Isolated or purified strain of human coronavirus associated with severe acute respiratory syndrome, characterized in that its genome has in the form of complementary DNA a serine codon at position 23220-23222 of the protein S gene or a glycine codon in position 25298-25300 of the ORF3 gene, and an alanine codon at position 7918-7920 of ORF1a or a serine codon at position 26857-26859 of the M protein gene, said positions being indicated with reference to Genbank sequence AY274119 .3.   2) isolated or purified strain of coronavirus according to claim 1, characterized in that the DNA equivalent of its genome has a sequence corresponding to the sequence SEQ ID NO: 1.   3) isolated or purified polynucleotide, characterized in that its sequence is that of the genome of the isolated coronavirus strain according to claim 1 or claim 2.   4) isolated or purified polynucleotide according to claim 3, characterized in that its sequence is SEQ ID NO: 1.   5) isolated or purified polynucleotide, characterized in that its hybrid sequence under conditions of high stringency with the sequence of the polynucleotide according to claim 3 or claim 4.   6) Fragment of the polynucleotide according to any one of claims 3 to 5, characterized in that it is obtainable, either by the use of restriction enzymes whose so-called recognition and cleavage are present in said polynucleotide according to any one of claims 3 to 5, or by amplification using oligonucleotide primers specific for said polynucleotide according to any one of claims 3 to 5, either by in vitro transcription or by chemical synthesis.   7) Fragment of the polynucleotide according to claim 6, characterized in that it is selected from the group consisting of: the cDNA corresponding to at least one open reading frame (ORF) chosen from: ORF 1a, ORF 1b, ORF-S, ORFE, ORF-M, ORF-N, ORF3, ORF4, ORF7 to ORF11, ORF13 and ORF14, and the cDNA corresponding to the 5 'or 3' non-coding ends of said polynucleotide.   8) Fragment according to claim 6, characterized in that it has a sequence selected from the group consisting of: the sequences SEQ ID NO: 2 and 4 representing the cDNA corresponding to the ORF-S which codes for protein S, the sequences SEQ ID NO: 13 and 15 representing the cDNA corresponding to the ORF-E which codes for the protein E, the sequences SEQ ID NO: 16 and 18 representing the cDNA corresponding to the ORF Which codes for the M protein, the sequences SEQ ID NO: 36 and 38 representing the ORF-N-encoding cDNA which encodes the N protein, the sequences representing the respective cDNAs respectively; : to ORFla and ORFIb (SEQ ID NO: 31), to ORF3 and ORF4 (SEQ ID NO: 7, 8), to ORFs 7 to 11 (SEQ ID NO: 19, 20), to ORF13 (SEQ ID NO 32), and to ORF14 (SEQ ID NO: 34), the sequences representing the cDNAs corresponding to the 5 '(SEQ ID NO: 39, 72) and 3' non-coding (SEQ ID NO: 40, 73) of the polynucleotide according to the endication 4.   9) Fragment according to claim 6, characterized in that it has a sequence selected from the group consisting of the sequences SEQ ID NO: 5, 6, and 41 to 54.   10) Fragment according to claim 6, characterized in that it has at least 15 consecutive bases or base pairs of the sequence of said polynucleotide including at least one of those located at position 7979, 16622, 19064, 23220, 24872, 25298 and 26857.   11) Fragment according to claim 10, characterized in that it includes at least one pair of bases or base pairs corresponding to the following positions: 7919 and 23220, 7919 and 25298, 16622 and 23220, 19064 and 23220, 16622 and 25298 , 19064 and 25298, 23220 and 24872, 23220 and 26857, 24872 and 25298, 25298 and 26857.   12) A pair of primers capable of amplifying a fragment of the genome of a SARS-associated coronavirus or its DNA equivalent, characterized in that it is selected from the group consisting of: ## EQU1 ## corresponding respectively to positions 28507 to 28522 (sense primer, SEQ ID NO: 60) and 28774 to 28759 (antisense primer, SEQ ID NO: 61) of the polynucleotide sequence according to claim 3 or claim 4, and - the pair of n 2 primers corresponding to positions 28375 to 28390 (sense primer, SEQ ID NO: 62) and 28702 to 28687 (antisense primer, SEQ ID NO: 63) of the polynucleotide sequence according to claim 3 or claim 4.   13) Probe capable of detecting the presence of the genome of a SARS-associated coronavirus or a fragment thereof, characterized in that it is selected from the group consisting of: fragments according to any one of Claims 6 to 11 and fragments corresponding to the following positions of the polynucleotide sequence according to Claim 3 or Claim 4: 28561 to 28586, 28588 to 28608, 28541 to 28563 and 28565 to 28589 (SEQ ID NOS: 64 to 67). ).   14) A chip or a DNA or RNA filter, characterized in that it comprises at least one polynucleotide or one of its fragments as defined in any one of Claims 3 to 11.   15) Cloning vector and / or recombinant expression, characterized in that it comprises a fragment according to any one of claims 6 to 11.   16) Recombinant vector according to claim 15, characterized in that it comprises the fragment of sequence SEQ ID NO: 4 and that it is included in a bacterial strain which has been deposited under the name I-3059, on June 20, 2003. , at the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15.   17) Recombinant vector according to claim 15, characterized in that it comprises the fragment of sequence SEQ ID NO: 5 and that it is included in a bacterial strain which has been deposited under the name I-3020, the 12 May 2003, at the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15.   18) Recombinant vector according to claim 15, characterized in that it comprises the fragment of sequence SEQ ID NO: 6 and that it is included in a bacterial strain which has been deposited under the name I-3019, on May 12, 2003 , at 2862981 63 the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15.   19) Recombinant vector according to claim 15, characterized in that it comprises a fragment of sequence SEQ ID NO: 8 and that it is included in a bacterial strain which has been deposited as I-3126, le, November 13, 2003, at the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15.   20) Recombinant vector according to claim 15, characterized in that it comprises the fragment of sequence SEQ ID NO: 15 and that it is included in a bacterial strain which has been deposited as I-3046, May 28, 2003 , at the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15.   21) Recombinant vector according to claim 15, characterized in that it comprises a fragment of sequence SEQ ID NO: 18 and that it is included in a bacterial strain which has been deposited as I-3047, May 28, 2003 , at the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15.   22) Recombinant vector according to claim 15, characterized in that it comprises a fragment of sequence SEQ ID NO: 20 and that it is included in a bacterial strain which has been deposited under the name I-3125, on November 13, 2003 , at the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15.   23) Recombinant vector according to claim 15, characterized in that it comprises an insert of sequence SEQ ID NO: 38 and that it is included in a bacterial strain which was deposited under the name I-3048, on June 5, 2003, from the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15.   24. Recombinant vector according to claim 15, characterized in that it comprises a fragment of sequence SEQ ID NO: 39 and that it is included in a bacterial strain which has been deposited under the name I-3124, on November 7, 2003. , at the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15.   25) Recombinant vector according to claim 15, characterized in that it comprises a fragment of sequence SEQ ID NO: 40 and that it is included in a bacterial strain which was deposited under the number I-3123 on November 7, 2003, from the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15.   26. Recombinant expression vector according to claim 15, characterized in that it contains a cDNA fragment selected from the group consisting of: a cDNA fragment coding for a C-terminal fusion of the N protein ( SEQ ID NO: 37) with a polyhistidine tag, - a cDNA fragment coding for a C-terminal fusion of the fragment corresponding to the positions 475 to 1193 of the amino acid sequence of the protein S (SEQ ID NO: 3) with a polyhistidine tag, a cDNA fragment encoding a C-terminal fusion of the fragment corresponding to positions 14 to 1193 of the amino acid sequence of protein S (SEQ ID NO: 3) with a polyhistidine tag, - a fragment cDNA encoding an N-terminal fusion of the N protein (SEQ ID NO: 37) with a polyhistidine tag; - a cDNA fragment encoding an N-terminal fusion of the fragment corresponding to positions 475 to 1193 of the amino acid sequence of protein S (SEQ ID NO: 3) with a polyhistidine tag, and - a cDNA fragment encoding an N-terminal fusion of the fragment corresponding to positions 14 to 1193 of the amino acid sequence of protein S (SEQ ID NO: 3 ) with a polyhistidine label.   27) recombinant expression vector according to claim 26, characterized in that it is included in a bacterial strain which was deposited under the number I-3117, on October 23, 2003, with the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15.   28) recombinant expression vector according to claim 26, characterized in that it is included in a bacterial strain which was deposited under the number I-3118, on October 23, 2003, from the National Collection of Cultures of Microorganisms, 25 rue du Docteur Roux, 75724 Paris Cedex 15.   29) cDNA library, characterized in that it comprises fragments according to any one of claims 6 to 11.   30) Vector-modified cells according to any one of claims 15 to 28 or a library according to claim 29.   31) Protein or peptide isolated or purified, characterized in that it is encoded by the polynucleotide according to any one of claims 3 to 5 or a fragment thereof according to any one of claims 6 to 11.   32) Isolated or purified protein according to claim 31, characterized in that it is selected from the group consisting of: protein S of sequence SEQ ID NO: 3, protein E of sequence SEQ ID NO: 14, the M protein of sequence SEQ ID NO: 17, the N protein of sequence SEQ ID NO: 37, and the proteins encoded by the ORFs: ORF 1a, ORF 1b, ORF3, ORF4 and ORF7 to ORF11, ORF13 and sequence ORF14 respectively, SEQ ID NO: 74, 75, 10, 12, 22, 24, 26, 28, 30, 33 and 35.   33) An isolated or purified peptide according to claim 31, characterized in that it is selected from the group consisting of: a) peptides corresponding to positions 14 to 1193 and 475 to 1193 of the amino acid sequence of protein S b) the peptides corresponding to positions 2 to 14 (SEQ ID NO: 69) and 100 to 221 of the amino acid sequence of protein M; and c) the peptides corresponding to positions 1 to 12 (SEQ ID NO: 70) and 53 to 76 (SEQ ID NO: 71) of the amino acid sequence of protein E; and d) peptides from 5 to 50 consecutive amino acids, preferably from 10 to 30 amino acids, included or partially or totally overlapping the sequence of the peptides as defined in a), b) or c).   34. Peptide according to claim 31, characterized in that it consists of 7 to 50 consecutive amino acids encoded by a fragment as defined in claim 10, which peptide is selected from the group consisting of: a peptide comprising alanine located at position 2252 of the amino acid sequence of the protein encoded by ORF 1a, - a peptide comprising serine located at position 577 of the amino acid sequence of protein S, - a peptide comprising glycine at position 11 of the amino acid sequence of the protein encoded by ORF3, and - a peptide comprising serine at position 154 of the amino acid sequence of protein M. 35) Antibody or fragment of monoclonal or polyclonal antibody , obtainable by immunization of an animal with a recombinant vector according to any one of claims 15 to 28, a library according to claim 29, or a protein or a peptide according to any one of claims 31 to 34, characterized in that it binds with at least one of the proteins encoded by the genome of the strain isolated or purified coronavirus, as defined in claim 31.   36) Protein or peptide chip or filter, characterized in that it comprises a protein or a peptide according to any one of claims 31 to 34 or an antibody or an antibody fragment according to claim 35.   37) SARS-associated coronavirus detecting reagent, characterized in that it is selected from the group consisting of: (a) a primer pair according to claim 12, a probe according to claim 13, or a chip or DNA or RNA filter according to claim 14, (b) a recombinant vector according to any one of claims 15 to 28 or a modified cell according to claim 30, (c) an isolated strain of coronavirus according to claim 1 or claim 2 or a polynucleotide according to any one of claims 3 to 5, (d) a protein or a peptide according to any one of claims 31 to 34, (e) an antibody or an antibody fragment according to claim 35, and (f) a protein or peptide chip or filter according to claim 36.   38) Use of a product selected from the group consisting of: a pair of primers according to claim 12, a probe according to claim 13, a chip or a DNA or RNA filter according to claim 14, a recombinant vector according to any one of claims 15 to 28, a modified cell according to claim 30, an isolated coronavirus strain according to claim 1 or claim 2, a polynucleotide according to any one of claims 3 to 5 for the preparation of a reagent for detecting and possibly genotyping a coronavirus associated with SARS.   39) A method for detecting a SARS-associated coronavirus from a biological sample, which method is characterized in that it comprises at least: (a) extracting nucleic acids present in said biological sample, (b) amplifying a fragment of the ORF-N by RT-PCR using a primer pair according to claim 12, and (c) detecting by any appropriate means the amplifications obtained in (b).   40. Method according to claim 39, characterized in that the step (c) of detection is carried out using at least one probe corresponding to positions 28561 to 28586, 28588 to 28608, 28541 to 28563 and 28565 to 28589 of the polynucleotide sequence according to any one of claims 3 to 5.   41) kit or kit for detecting a coronavirus associated with SARS, characterized in that it comprises at least one reagent selected from the group consisting of: a pair of primers according to claim 12, a probe according to claim 13, a DNA or RNA chip or filter according to claim 14, a recombinant vector according to any one of claims 15 to 28, a modified cell according to claim 30, an isolated strain of coronavirus according to claim 1 or claim 2 and a polynucleotide according to any one of claims 3 to 5.