Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/20011—Rhabdoviridae
  • C12N2760/20111—Lyssavirus, e.g. rabies virus
  • C12N2760/20122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• the present invention relates to a method for detecting and identifying Lyssavirus infections, to the cloning and to the expression of genes coding for peptides and / or fragments of peptides of Lyssavirus Mokola, to a vaccine against the Mokola virus and / or all of the Lyssaviruses as well as its process for obtaining by genetic engineering.
• Lyssavirus strains isolated from bats in northern Europe and Spain are currently being classified. These viruses, although currently only isolated from bats, pose the problem of protecting the human population and other animal species, and in particular the problem of vaccine protection against these viruses.
• the Mokola virus is also of concern, since it has been made responsible in Africa for isolated cases of fatal rabiform encephalitis in many species, including humans, and especially for an epidemic in domestic Malawi carnivores, including a dog. who was vaccinated against rabies.
• a number of documents describe nucleotide sequences of the rabies virus and the vaccines derived therefrom.
• the French patent THE WISTAR INSTITUTE 2,515,685 proposes a complementary synthetic DNA which codes for the glycoprotein of the rabies virus of strain ERA, which is defined by its nucleotide sequence, its initiation codon ATG and its termination codon TGA, which is a copy of the mRNA of said glycoprotein and which is a single-stranded cDNA.
• fragments of polypeptides of this cDNA between two cleavage sites cannot be greater than 50 amino acids.
• the deduced amino acid sequence of the glycoprotein comprises 524 amino acids with a signal peptide of 19 non-polar amino acids preceding the amino-terminal lysine residue and cleaved in the mature protein.
• the cDNA of the rabies virus and the mRNA of the glycoprotein can be used to transform a bacterium in order to produce a polypeptide in sufficient quantities to carry out immunization.
• European Patent Application TRANSGENE 94 887 relates to a vector, such as a phage or a plasmid, for the expression of an antigenic protein of rabies, and more particularly the glycoprotein, which comprises at least one efficient DNA sequence which code for said protein and a promoter for the expression of this sequence in a bacterium, the coding effective DNA sequence possibly being a total effective DNA sequence or a partial sequence lying between two determined cleavage sites.
• This vector is used to transform or transfect - depending on whether it is a plasmid or a phage - a bacterium by culture from which the antigenic protein of the desired rabies is obtained, which is challenged negated by its amino acid sequence and which is used as an active component of a rabies vaccine.
• the claims of this Application essentially relate to the partial polynucleotide sequence 71-1 421 which codes for the nucleoprotein, to the partial sequence 1 514-2404 which codes for the protein Ml, to the partial sequence 2 496-3 102 which codes for protein M2, on the partial sequence 5 417-5 500 which codes for the N-terminal part of protein L.
• nucleotide sequences and their fragments and others suggested in this Application inserted into vectors, modify these vectors which, when introduced into an appropriate host cell, oblige it to transcribe and translate the DNA sequences. the above to produce the corresponding proteins which can then be isolated from cell extracts of said host cell.
• This Application also covers recombinant DNAs which contain one of the abovementioned inserts placed under the control of a promoter derived from the genome of the SV40 virus, which are vectors which can be used to transform eukaryotic cells (such as Vero cells) and produce proteins endowed with immunological properties, the promoter being able, more generally, to be a viral or eukaryotic promoter recognized by the polymerases of the selected cells and which additionally comprises suitable polyadenylation sites downstream of the insert.
• This Patent Application men Furthermore, one of the major advantages of the invention is to provide DNA sequences derived from the genomic RNA of the rabies virus which, unlike the genome itself, can be isolated under a form devoid of nucleoproteins.
• the cDNAs according to this Patent Application can be used as probes, for example for the detection of the presence in a biological fluid, of the rabies virus, by hybridization.
• N, M1, M2 polypeptides as defined in this Patent Application and the aforementioned Articles, by their peptide structures, can be used to produce corresponding antibodies which can themselves be used for the in vitro diagnosis of the presence of polypeptides viral in a biological fluid, the protein M1 being of very particular interest, in particular as an immunogenic composition in association with a vehicle used in the production of vaccines.
• the article in the name of O. POCH et al. published in Biochemistry, 1988, 70, 1019-1029 describes cDNA fragments of the genome of an avirulent strain (AVO1) of the rabies virus.
• AVO1 avirulent strain
• the 3,386 nucleotide sequence from the 3 'end covers the genes encoding the leader RNA, nucleoprotein N, phosphoprotein M1 and the matrix protein M2, as well as the intergenic regions.
• Comparison of the AVO1 sequence with that of other strains of the rabies virus reveals significant conservation both at the nucleotide and amino acid level.
• rabies genome Comparison of the rabies genome with those of other non-segmented negative single-stranded RNA viruses (rhabdovirus and paramyxovirus) indicates that the signals for initiation and stop of transcription, located at the ends of each gene coding for a protein, and the regions phosphoprotein and matrix proteins which could be involved in the transcription process maintain a similar overall structure.
• the Mokola virus is a so-called "related" rabies virus: rabies vaccines provide very little protection against infection by the Mokola virus.
• mice vaccinated with the three vaccines available on the market (PV strain (Pasteur); PM strain (Mérieux); Flury LEP strain (Behring)) do not withstand a test by the virus Mokola.
• their serum contains only few antibodies capable of neutralizing the Mokola virus in vitro, and their lymphocytes exhibit low cytotoxicity with respect to target cells infected with the Mokola virus.
• This hybridization method is used to detect rabid transcripts in the brain.
• 32 p-labeled cDNA probes from the genome of the rabies strain PV (serotype 1) are used to identify very small amounts of specific viral RNA.
• the purified viral RNA is obtained after phenolic extraction.
• the RNA is fixed on nylon membranes and hybrid with a pool of M13 inserts complementary to 200-400 nucleotides of each rabies gene and each mRNA.
• the labeled hybridized probes are detected by autoradiography. Hybridization was observed with brain samples from animals inoculated with vulpine strains of rabies virus (serotype 1). A positive response was obtained for amounts of total RNA of 80 ng.
• Detection of viral transcripts remained possible on brains taken a week after the animal's death. A total correlation is observed in comparison with other techniques such as the detection of rabies antigens by the use of a fluorescent anti-rabies antibody or the isolation of the virus on murine neuroblastome cells.
• the purpose of the present invention is to provide a specific vaccine against the Mokola virus obtained by expressing viral antigens mainly involved in the immune response as well as a polyvalent vaccine directed against all the Lyssavirus serotypes, obtaining by engineering
• the advantage of genetics is that it solves the problems of virus production and provide a specific diagnostic agent that is very sensitive to Lyssavirus infections.
• the subject of the present invention is a process for the rapid detection and / or identification of small amounts of Lyssavirus present in a biological sample, characterized in that said sample suitably treated to extract the viral RNA and / or the transcription products of the Lyssavirus possibly present is:
• the cDNA sequence is brought into contact with a pair of suitable Lyssavirus primers to amplify at least one fragment of said cDNA, one of said primers being different from that of step (1) and the other primer being identical to or different from that of step (1);
• the PCR method is notably described by SAIKI et al. in Science, 1988, 239, 487 as well as in European Patent Applications CETUS No 200 362 and 201 184.
• the method of the present invention unexpectedly allows rapid detection (less than 12 hours) of a Lyssavirus infection from a saliva or organ sample and then identify the Lyssavirus.
• the primers are chosen from the group which comprises the primers specific for a strain, for a serotype of Lyssavirus, the specific primers specific for a Lyssavirus serotype and the primers consisting of a conserved sequence of a gene common to all Lyssaviruses and hereinafter called polyvalent primers.
• the primers for diagnosis or detection of a Lyssavirus (Lyssavirus infection: response of the Yes / No type) and the primers for differential diagnosis or typing ( Lyssavirus infection: details on type).
• the primer is a 3 'rabies primer, located in position 1-18 of the rabies and Mokola genomes.
• the primer is a rabies primer M2, located in position 2901-2918 of the rabies genome.
• the primer is a rabies primer consisting of 23 nucleotides, located in position 4665-4687 of the rabies genome and in position 4675-4697 of the Mokola genome, hereinafter called primer G .
• the primer is a rabies primer consisting of 24 nucleotides, located in position 5520-5543 of the rabies genome and in position 5545-5568 of the Mokola genome, hereinafter called primer L .
• the primer is a Mokola or rabic primer consisting of 18 nucleotides located in position 587-605 of the rabies and Mokola genomes, hereinafter called the Na primer.
• the primer is a Mokola or rabic primer consisting of 16 localized nucleotides in position 1013-1029 of the rabies and Mokola genomes, below called primer Nb.
• the detection of the amplified nucleotide sequence is carried out by hybridization using a probe or a battery of probes suitable for the various Lyssaviruses.
• the detection of the amplified nucleotide sequence is carried out by cleavage of said sequence with at least one battery of suitable restriction enzymes followed by separation by electrophoresis of the fragments obtained.
• the battery of enzymes comprises BamH I, Hind II, Hind III, Pst I.
• the battery of enzymes comprises Rsa I, Taq I, BstX I, Fnu4H I.
• the rapid detection method, according to the invention of small amounts of Lyssavirus in a sample, has the advantage of making it possible to diagnose Lyssavirus infection on early infections or on saliva samples taken from domestic animals suspected of being responsible for human contamination, several days before their death, contrary to current practice; indeed, for example, the pair of primers, Na primer and Nb primer as defined in the invention, located on the gene N and not far apart from one another (400 bp) allows the detection (Yes / No response) of a Lyssavirus even present in very small quantities, even if the sample is very degraded. Indeed, the sensitivity of the usual techniques is relatively low compared to the method according to the invention, which is rapid and allows a diagnosis to be made in less than 12 hours.
• the method according to the invention allows identification and typing of the Lyssavirus involved in the infection to be detected.
• the pair of primers, rabies primer G and rabies primer L as defined above, associated with a battery of appropriate restriction enzymes makes it possible to type both the different strains of rabies virus, the virus Mokola, as strains of Lyssavirus isolated from bats; the two aforementioned primers are chosen so as to correspond to conserved zones in Lyssaviruses, one in the distal part of the G gene, the other in the proximal part of the L gene, to allow the amplification of the region which separates, around 860 nucleotides (pseudogen psi), on all rabies strains as well as on Mokola.
• the amplified band is detected either by hybridization with a more or less specific strain-specific probe, or by hydrolysis in the presence of more or less strain-specific restriction enzymes.
• a direct sequencing technique can be applied directly to the piece of agarose containing the amplified band.
• the present invention also relates to the specific sequences of the Mokola virus, which sequences have made it possible to highlight the conserved areas and the variable areas in the genus Lyssa. virus and by comparison between the rabies and Mokola genomes, to assess the variability of each genomic region and to develop the detection and / or identification method of Lyssavirus above.
• the nucleotide sequence of the cDNA of the genomic RNA of the Mokola virus is characterized in that it comprises approximately 12,000 nucleotides.
• Said genomic RNA cDNA sequence is further characterized in that the 3 'and 5' ends are complementary, in that the 12 nucleotides of the 5 'end are identical to those of the PV strain of rabies virus, and in that it successively presents from 3 'to 5' the gene coding for the "leader" RNA then the genes coding for the proteins N, M1, M2, G and L.
• sequence of the cDNA of the genomic RNA of the Mokola virus comprises the sequence in nucleotides and the deduced sequence in amino acids as follows (I):
• Val-Ile-Val-Asp-Ile-Val-Arg-Thr-Asn-Val-Glu-Gly-Asn-Trp- GTG ATC GTC GAT ATA GTT CGC ACT AAC GTT GAG GGG AAT TGG 430
• TTC CAT AAA AAC TTT GAA GGG GAG ATT AAG AGA ATG TTT GAG 892
• GAG GAG GCC AGA GTA GAG GCT TCG CTC GCT GAT GAC GGG ACT 1228
• AGAACAATTA CCGCAACGGT GCCCGCTTTC AGCACAATAC ATATAAGCTA 3241 ACCACTGGTT TGTCTTCCTA TTCAGGGTCG AGCGAAAACG 3291 Met-Asn-Ile-Pro-
• GCT GAC ATC TTA CCC TCT AAG GGA TGT CTG AAA GTC GGG CAA 4431
• TTATAAAGAG TTGCCTTCTA AAATGGGCAC TCTATAGAGC CTTCAATCTT 5193 TTTGAGGTGC GGCAATATTA GCTTGAAATA ACCTTAAGGT CTAATTTCTC 5243
• TCT TTC CTC TCA AGG GGC CCT CTT AAG GGG TAC TTA GGA TCT 8946
• the subject of the invention is also, fragments of said sequence coding for one of the peptides and / or for a peptide fragment of the Mokola virus as well as fragments of said non-coding sequence corresponding to a variable region of the Mokola genome, that is to say ie not conserved of a gene common to all Lyssaviruses.
• coding fragments includes inter alia:
• N corresponds to nucleotides 71-1420 of said cDNA sequence
• non-coding fragments includes inter alia a fragment which corresponds to nucleotides 4897-5442 of said sequence, which fragment corresponds to the psi rabies pseudogen.
• the subject of the present invention is also the sequence of the genomic RNA of the Mokola virus, characterized in that it comprises approximately 12,000 nucleotides, in that it is a non-segmented and non-polyadenylated negative single-stranded RNA, in that it successively presents from 3 'to 5' the gene coding for the "leader" RNA then the genes coding for the nucleoprotein N, the phosphoprotein M1, the matrix protein M2, the gly co-protein G and protein polymerase L and in that said genome is always associated with nucleoprotein N.
• the present invention also relates to the transcripts of the Mokola virus, characterized in that they consist of 5 successive monocistronic fragments coding from the 3 ′ end for the proteins N, M1, M2, G and L, to know :
• the present invention also relates to cDNA clones of the genomic RNA of the Mokola virus, characterized in that they correspond to the entire genome, from the 3 ′ end to the 5 ′ end, namely :
• pMD10 a fragment which measures 4,150 nucleotides, hereinafter called pMD10 and corresponding to the sequence coding for RNA "leader" for nucleoprotein N, protein M1, protein M2 and a fragment of the sequence coding for protein G ;
• pMA10 a fragment which measures 2,850 nucleotides
• pMA10 a fragment which measures 2,850 nucleotides
• pM7 a fragment which measures 2,850 nucleotides
• pMB5 a fragment of approximately 3,300 nucleotides, called pMB5 and corresponding to a fragment of the sequence coding for the protein L;
• pM12 a fragment of approximately 2800 nucleotides, hereafter called pM12, corresponding to a fragment of the sequence coding for the protein L;
• pMR15a a fragment of approximately 700 nucleotides, hereinafter called pMR15a and corresponding to a fragment of the sequence coding for the protein L as well as at the 5 'non-transcribed end of the genome;
• fragments taken separately, each have an ability to hybridize specifically to an RNA fragment originating from the transcription or replication of the Mokola genome or to a cDNA fragment.
• the pM7 clone was deposited under the number 1-847 dated March 22, 1989 with the National Collection of cultures of microorganisms held by the Pasteur Institute.
• the pMB5 clone was deposited under the number 1-848 dated March 22, 1989 with the National Collection of microorganism cultures held by the Pasteur Institute.
• the pM12 clone was deposited under the number 1-849 dated March 22, 1989 with the National Collection of cultures of microorganisms held by the Pasteur Institute.
• the clone pMR15a was deposited under the number 1-850 dated March 22, 1989 with the National Collection of cultures of microorganisms held by the Pasteur Institute.
• the present invention also relates to nucleotide probes, characterized in that they consist of a nucleotide sequence as defined above or a fragment thereof, labeled with the aid of a marker such as a radioactive isotope, a suitable enzyme or a fluorochrome.
• probes According to one embodiment of said probes, mention may be made of the sequence 4675-5568 or a fragment thereof and in particular the fragment 4897-5442 or their complementary strands.
• Such probes are specific for the Mokola virus.
• the present invention also relates to peptides or peptide fragments, characterized in that they are encoded by at least one fragment as defined above or a fragment portion or a combination of several fragments as defined above.
• said peptide is encoded by the nucleoprotein N gene and has an amino acid sequence which corresponds to formula II below:
• said peptide is coded by the gene for the protein M1 and has an amino acid sequence which corresponds to formula III below: Met-Ser-Lys-Asp-Leu-Val-His- Pro-Ser-Leu-Ile-Arg-Ala-Gly- Ile-Val-Glu-Leu-Glu-Met-Ala-Glu-Glu-Thr-Thr-Asp-Leu-Ile- Asn-Arg-Thr-Ile- Glu-Ser-Asn-Gln-Ala-His-Leu-Gln-Gly-Glu- Pro-Leu-Tyr-Val-Asp-Ser-Leu-Pro-Glu-Asp-Met-Ser-Arg-Leu- Arg- Ile-Glu-Asp-Lys-Ser-Arg-Arg-Thr-Lys-Thr-Glu-Glu-Glu-Glu-Arg-Asp-Glu-Gly-Ser-Ser-Glu-Glu-Asp-Glu-Asp
• said peptide is encoded by the protein M2 gene and has an amino acid composition which corresponds to formula IV below:
• said peptide is coded by the glycoprotein G gene and is characterized by an amino acid sequence which corresponds to formula V below:
• said peptide is coded by the L protein gene and has one of the following amino acid sequences:
• peptides and / or peptide fragments in accordance with the invention are obtained by synthesis.
• the present invention also relates to a vector, characterized in that it contains at least one nucleotide sequence or a portion of this sequence as defined above.
• the present invention also relates to a vector for the expression of a peptide or of a peptide fragment or of a combination of fragments of peptides of the Mokola virus, characterized in that it is obtained by homologous recombination between a baculovirus. of wild strain and an appropriate shuttle vector comprising at least minus:
• said shuttle vector comprises:
• the gene or a fragment of the gene for the glycoprotein G of the Mokola virus is inserted at the level of the polylinker, so as to obtain an expression vector said to be charged with said glycoprotein, under the control of the promoter of the polyhedrin gene.
• said expression vector was deposited under the number 1-851 dated March 22, 1989 with the National Collection of Cultures of Microorganisms held by the Institut Pasteur.
• the gene or a fragment of the nucleoprotein N gene is inserted at the level of the polylinker, so as to obtain an expression vector said to be charged with said nucleoprotein, under the control of the gene promoter polyhedrin.
• the process of expression of at least one peptide, a fragment of a peptide or a combination of frag peptides of the Mokola virus implements an expression vector in accordance with the invention, in appropriate lepidopteran cells, in particular the caterpillar Spodoptera frugiperda, Sf9 in the presence of wild-type baculovirus, for obtaining recombinant baculoviruses expressing the desired polypeptide.
• recombinant baculoviruses will be based on the morphology of the ranges obtained. Indeed, the presence of polyhedrin gives the beaches of wild virus a refractive aspect in light microscopy when they are illuminated by epiluminescence. Conversely, the dull appearance of the recombinant virus ranges, due to the absence of crystal, makes it easy to distinguish them.
• the expression level can be extremely high, of the order of 1 to 10 mg per liter of culture;
• polyhedrin is a late gene expressing itself after viral replication and the formation of nucleocapsides have taken place, even allows proteins toxic for the multiplication of the virus to be expressed.
• the present invention also relates to a vaccine against the Mokola virus, for human and / or veterinary use, characterized in that it comprises at least one peptide and / or a peptide fragment in accordance with the invention, optionally associated with at least one pharmaceutically acceptable vehicle.
• a vaccine against the Mokola virus for human and / or veterinary use, characterized in that it comprises at least one peptide and / or a peptide fragment in accordance with the invention, optionally associated with at least one pharmaceutically acceptable vehicle.
• said vaccine comprises the glycoprotein G and / or a fragment thereof and / or the nucleoprotein N or a fragment thereof.
• the combination of these two viral peptides has the following advantages: they intervene at different levels of the immune response; in fact the glycoprotein G preferentially induces the formation of neutralizing antibodies whereas the nucleoprotein N intervenes especially in cell-mediated immunity.
• the present invention also relates to a polyvalent Lyssavirus vaccine, characterized in that it comprises at least one peptide and / or peptide fragment in accordance with the invention, optionally associated with at least one peptide and / or a peptide fragment of at least one other Lyssavirus serotype.
• Lyssaviruses of serotype 1 rabies virus
• Lyssaviruses of serotype 2 Lagos bat virus
• Lyssaviruses of serotype 4 Duvenhage virus
• Lyssaviruses isolated on European bats close to serotype 4.
• said peptides and / or peptide fragments are advantageously associated with an appropriate support and / or an acceptable adjuvant, in particular the conventional adjuvants of human and veterinary vaccines.
• the present invention also relates to monoclonal antibodies specific for the peptides and / or peptide fragments of the Mokola virus, characterized in that they result from the immunization of mammals, in particular rodents, and more particularly of mice, by peptides and / or peptide fragments in accordance with the invention.
• the present invention also relates to an immunological method for detecting anti antibodies peptides and / or peptide fragments of the Mokola virus, which consists in detecting anti-Mokola antibodies possibly present in a biological sample using a peptide or a peptide fragment in accordance with the invention, by bringing together said biological sample with said peptide (s) or peptide fragment (s), to which the anti-Mokola antibodies bind if such antibodies are present in the biological sample to be analyzed, the reading of the result being revealed by an appropriate means, in particular EIA , RIA, fluorescence.
• an appropriate means in particular EIA , RIA, fluorescence.
• said peptides or peptide fragments are fixed on an appropriate solid support.
• Said immunological method can advantageously be of direct type, of indirect type or implement a sandwich or bi-site method.
• the revelation is carried out by means of a second antibody directed against the antibody to be assayed and appropriately labeled.
• This process allows the serum antibody titration and in particular makes it possible to verify the seroconversion of vaccinated individuals or to carry out serological surveys for epidemiological purposes.
• the subject of the present invention is also a method of rapid and specific detection of the Mokola virus which consists in detecting a Mokola virus, possibly present in a biological sample using at least one nucleotide probe according to the invention, by putting in the presence of said biological sample treated appropriately with said at least one nucleotide probe, to which / to which the genomic RNA and / or the transcripts of the Mokola virus binds, if such pro duits are present in the sample, the reading of the result being revealed by an appropriate means.
• the subject of the present invention is also a ready-to-use kit for implementing the method according to the invention for detecting and / or identifying at least one Lyssavirus, characterized in that it further comprises useful quantities of buffers and reagents suitable for carrying out said detection, appropriate doses of at least two suitable primers, appropriate doses of at least one nucleotide probe and appropriate doses of at least one enzyme restriction.
• the subject of the present invention is also a ready-to-use kit for implementing the method for determining in a biological sample, anti-Mokola antibodies, characterized in that it comprises at least:
• the peptide is glycoprotein G or a fragment thereof, fixed on an appropriate solid support.
• the peptide is nucleoprotein N or a fragment thereof, fixed on an appropriate solid support.
• This kit can also comprise appropriate doses of a second antibody directed against the antibody to be assayed labeled with the aid of an enzyme, a fluorescent substance or a radioactive isotope.
• the present invention further relates to a ready-to-use kit for implementing the method for detecting a Mokola virus according to the invention, characterized in that it comprises at least: - appropriate doses of at least one probe and / or fragment of nucleotide probe according to the invention; and
• the invention also comprises other provisions, which will emerge from the description which follows, which refers to examples of implementation of the invention.
• Example 1 Cloning and sequencing of DNA complementary to the genomic RNA of the Mokola virus.
• the Mokola strain studied was isolated from a cat in Moscow.
• the virions are purified from the lysis plaques obtained on a culture of CER cells.
• the Mokola virus thus selected is cultured on BHK-21 cells and purified according to the method described by WIKTOR et al. (J. Virol., 1977, 21, 626-635) modified.
• the purified virions are incubated with 100 ⁇ g / ml of proteinase K (Merck) in 1.5% SDS, 100 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA for 30 min at 37 ° C, followed by two phenol-chloroform extractions (vol / vol) and precipitation with ethanol.
• proteinase K Merck
• the first strand of cDNA is synthesized in the presence of 1 ⁇ g of genomic RNA using a molar ratio 10 times greater in the presence of one octodecameric primer at a time (the 3 'primer, located in position 1-18 or the primer M2, located in position 2,901-2,918 on the rabies genome, for example) in the presence of 50 units of reverse transcriptase, in a buffer containing 50 mM of Tris HCl pH 8.3, 8 mM of MgCl 2 , 100 mM KCl, 0.8 mM dATP, 0.8 mM dGTP, 0.3 mM dCTP, 0.3 mM dTTP, 0.2 ⁇ M 32 P dCTP, 0.2 ⁇ M 32 P dTTP, 30 U / ⁇ l RNasin for 2 hours at 42 ° C.
• the double stranded cDNA is prepared according to the GUBLER and HOFFMAN method and separated according to its size on a Biogel A-50 m column.
• fractions (approximately 15 to 20 ng of cDNA) corresponding to the largest cDNAs are inserted at the Pst I site of the plasmid pBR322 using the dC / dG extension method.
• the cDNA library obtained with the 3 'primer makes it possible to select the clone pMA10 with Mokola probes originating from the first cloning. From this, the pMD10 clone is selected.
• the inserts pMR15a and pMD10 have been sequenced and reach the 5 'and 3' ends of the genomic RNA respectively, proving that the five clones mentioned above are sufficient to cover the entire genome of the Mokola virus.
• Figure 2 compares the 3 'and 5' ends of the genome of the Mokola virus with those of the PV strain of the rabies genome.
• the complementary nucleotides between the two ends are specified using long vertical lines.
• the consensus sequences specifying the start of the N gene mRNA, the stop of the L gene mRNA and the start signal of the N protein are indicated.
• the nucleotides of the 3 'and 5' genomic ends of the Mokola virus which are identical to those of the PV strain are specified using short vertical lines. The residues are numbered from the genomic ends.
• FIG. 3 shows the heterologous and homologous hybridization of rabies probes located on the genes
• cytoplasmic RNA obtained from BHK-21 cells, is collected 6, 12, 24 or 48 hours after infection with the Mokola virus (traces 1) or infection with the rabies virus (trace 2); trace 3 corresponding to a negative control (uninfected cells), then is subjected to electrophoresis on 1.2% agarose gel - formaldehyde. The separated RNAs are deposited on nylon and hybrid membranes with a probe labeled with
• 32 P consisting either of the sequence 377-1039 of the cDNA of the rabies genome (probe N: A), or of the sequence
• the cDNA probes are selected in accordance with FIG. 1 to characterize the transcripts in vivo during an infection with the Mokola virus of BHK-21 cells.
• Six different transcripts are observed by the Northern blot analysis, as visible in FIG. 4 which shows the mRNAs of the Mokola virus hybridizing with the cDNAs N, N + M1, M1 + M2, G and L of the genome of the Mokola virus .
• the total cytoplasmic RNA of Mokola virus obtained from infected BHK-21 cells, is denatured in the presence of 50% formamide and 15 ⁇ g samples are subjected to electrophoresis on 1% agarose gel containing formaldehyde. The separated RNA is deposited on nylon membranes with cDNA probes labeled with 32 P (N, M1 + M2, N + M1, G and L). The arrows indicate the positions of the 18 S and 28 S ribosomal RNA, revealed by staining with ethidium bromide and the identity of the different mRNAs is revealed by autoradiography.
• Example 3 Method for determining the glycoprotein G of Mokola virus in a preparation or a biological liquid in the presence of an anti-glycoprotein 6 antibody according to the invention conjugated with horseradish peroxidase.
• the suspension is clarified by centrifugation at 1500 xg for 30 min at 4 ° C.
• the clarified supernatants from each sample are distributed in duplicate in the wells of microtitration plates sensitized with anti-glycoprotein G antibodies in accordance with the invention (200 ⁇ l / well).
• the microplates are then incubated for one hour at 37 ° C. After repeated washing with PBS-Tween buffer, each well receives 200 ⁇ l of anti-glycoprotein G antibody conjugated with horseradish peroxidase.
• the microplates are then incubated for one hour at 37 ° C, then washed again.
• the viral antigen is then quantified by the appearance of a coloration, when the substrate of the peroxidase is added; the substrate is a mixture of o-phenylenediamine and hydrogen peroxide (200 ⁇ l / well).
• the microplates are left 20 min at room temperature to allow the staining to develop.
• the reaction is stopped by adding H 2 SO 4 N (50 ⁇ l / well).
• the coloration is measured with a spectrophotometer.
• Example 4 Method for determining the glycoprotein G of Mokola virus in the presence of an anti-glycoprotein 6 antibody according to the invention conjugated to fluorescein isothiocyanate.
• Cell cultures infected with the Mokola virus are fixed for 30 min in cold acetone. Staining is carried out by covering the slides for 30 min at 37 ° C. with anti-glycoprotein G antibodies of Mokola virus conjugated to fluorescein isothiocyanate. Evans blue (1/5000) is used as a counter-dye. The slides are washed by immersion in PBS at pH 7.6 for 5 min. A mounting medium at the glycerin is used and the slides are examined under an epifluorescence microscope.
• Example 5 Method for detecting and titrating anti-glycoprotein 6 antibodies of the Mokola virus, in the blood of vaccinated individuals, with peptides and / or fragments of peptides in accordance with the invention.
• This test is based on the use of a solid phase, on which the virus glycoprotein is attached, and of an enzyme conjugate, protein A of Staphylococeus aureus coupled with peroxidase.
• Two-fold dilutions are made of positive and negative control sera. Unknown sera or plasmas are diluted to 1/100. 100 ⁇ l of these different sera are deposited in each well.
• the adhesive film is removed; the contents of each plate are aspirated, three successive washings of the wells are carried out, then the plates are dried.
• the contents of the wells are aspirated, washed four times and then the plates are dried.
• 3.5 ⁇ l (approximately 0.5 picomoles) of cloned M13 matrix are placed in the presence of 1 ⁇ l (0.5 picomoles) of universal primer and 0.5 ⁇ l of a buffer comprising 10 mM Tris-HCl pH 7, 5, 10mM MgCl 2 , 50mM NaCl, ImM DTT.
• the mixture hermetically closed, is immersed in a bain-marie which is brought to a boil and then allowed to cool gradually on the bench. After 1/2 h, the water is at 40 ° C and the hybridization is done.
• reaction takes place for 20 min at + 37 ° C, then is stopped by heating for 3 min at + 65 ° C.
• the M13 matrix, copied by the polymerase from the universal primer is in double strand over a length depending on the amount of mononucleotides initially introduced into the medium. In addition, this length varies statistically from one clone to another.
• the synthetic reaction mixture (15 ⁇ l) is brought to a salt concentration suitable for the restriction enzyme chosen (MANIATIS et al., 1982), and the cut takes place for 1 h, at the appropriate temperature, in a final volume of 20 ⁇ l.
• An identical volume of a denaturing deposit buffer is then added. After thermal denaturation, the mixture is deposited on a 6% denaturing acrylamide-urea gel (0.5 mm thick). The migration lasts 1 hour at 1200 volts.
• the radioactive probe is identified by autoradiography and then cut out.
• This probe can then be used for direct hybridization on blots or for S1 nuclease mapping.
• the techniques we follow are classic, although slightly modified. Here are some details:
• Example 7 Detection of a Mokola virus RNA using a probe in accordance with the invention (RNA blots)
• a hybridization medium which includes:
• BSA bovine serum albumin
• Example 8 Method for rapid identification (typing) of small quantities of Lyssavirus.
• RNA from BHK21 fibroblastic cells or N2A murine neuronal cells infected with a Lyssavirus (classic Rabies strains Pasteur, PV, CVS, Av01, PM, ERA, wild rabies strains "Gabon dog", “Brazilian bat “,” European fox “, Mokola, etc ).
• primer G ", corresponding to an oligonucleotide long 23 nucleotides, of direction (+) and extending between positions 4665 and 4687 of the rage PV genome, and a DNA of sense (+) complementary to the genome is specifically initiated and synthesized as described in Example 1.
• the reaction medium is extracted with phenol, precipitated with ethanol, washed twice with 70% ethanol, then dried. It is then taken up in 50 ⁇ l comprising:
• the primer "L” corresponds to an oligonucleotide 24 nucleotides long, of direction (-) and extending between positions 5520 and 5543 of the PV rabies genome.
• the primer G hybridizes with the region 4675-4697 of the nucleotide sequence (I) above, which primer G has a homology of the order of 80% with the region 4675-4697 of the sequence (I) of the Mokola virus.
• Primer L hybridizes specifically with region 5545-5568 of the nucleotide sequence of formula (I) of the Mokola virus.
• reaction medium is placed in a 1.5 ml eppendorf tube, covered with 100 ⁇ l of mineral oil to avoid evaporation, and incubated in a "P.C.R. machine" subjecting him to the following stages:
• This step is carried out once, then it is repeated 40 times in succession, limiting the denaturation time to 2 min.
• the amplified cDNA band can be observed after migration of the reaction products on an appropriate agarose gel. After transfer, it can be characterized by means of an appropriate probe, originating from the cloning of the genome of the strain analyzed or of another strain of Lyssavirus.
• Figure 7 corresponds to an electrophoresis gel and has the following 16 traces:
• the amplified cDNA band can also be hydrolyzed by selected restriction enzymes capable of quickly differentiating:
• the other two enzymes are more group specific:
• BstX I only cuts PV, ERA and SAD; .
• Fnu4H I only cuts wild rabies strains and the Mokola virus.
• FIG. 8 shows that the amplified bands originating from the ERA and CVS strains are respectively and specifically cut by Bstx I and BamH I.
• Example 9 Method for rapid detection (Yes / No response) of small amounts of Lyssavirus.
• the primers Na and Nb as defined in the invention allow, for example, the amplification of the 587-1029 region of the nucleotide sequence of formula (I) of the Mokola virus which is a conserved region of a Lyssavirus; this method of rapid detection of small amounts of Lyssavirus in a sample in accordance with the invention has the advantage of making it possible to make a diagnosis of infection with Lyssavirus on early infections or on saliva samples taken from suspect domestic animals human contamination, several days before their death, contrary to current practice; indeed, the primers Na and Nb as defined in the invention, located on the N gene and not far apart from one another (400 bp) allow the detection (Yes / No response) of a Lyssavirus even present in very small quantities, even if the sample is very degraded. Indeed, the sensitivity of the usual techniques is relatively low compared to the method according to the invention, which is rapid and allows a diagnosis to be made in less than 12 hours.
• the inserts pMD10 and pMA10 derived from the cloning of the Mokola genome, each cover half of the glycoprotein G gene. Thanks to the restriction site BglI which they have in common, they can be joined in a single insert pM7 covering the 6,830 nucleotides 3 'of the genome. from this, we isolate the cDNA from the glyco gene protein G by a double digestion combining the restriction enzymes FnuDII and Sspl, which is inserted between the promoter region 5 ′ of the baculovirus polyhedrin gene and its region 3 ′ corresponding in particular to the polyadenylation signal. This construction is carried out in a vector pUC, to allow its multiplication.
• Figure 5b corresponds to a negative control.
• the glycoprotein was also clearly localized on the surface of the cells infected with the recombinant virus, by means of fluorescent monoclonal antibodies specific for the glycoprotein of the Mokola virus (FIG. 6).
• traces 1, 2 and 3 correspond to Spodoptera frugiperda cells after infection with the recombinant baculovirus expressing the glycoprotein G of the Mokola virus: the arrow shows the location of the glycoprotein G on the gel; Trace T corresponds to a negative control.
• Example 11 Demonstration of the immunogenic power of the glycoprotein of the Mokola virus (G. MOK).
• FIG. 9 shows that the mortality curves of unvaccinated (NV) mice and vaccinated with SF9 cells infected with NPEV del are little different. SF9 cells infected with NPEV del therefore do not give any non-specific protection to mice.
• FIG. 10 shows that a vaccine dose of 100,000 SF9-G.MOK cells is sufficient to protect mice and 10 6 SF9-G.MOK cells are sufficient to obtain a protection index greater than 2.5.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Medicinal Chemistry (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Virology (AREA)
• Genetics & Genomics (AREA)
• General Health & Medical Sciences (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Gastroenterology & Hepatology (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Peptides Or Proteins (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=9380140

Family Applications (1)

Country Status (5)

Families Citing this family (3)

Family Cites Families (4)

• 1989
  • 1989-03-29 FR FR898904052A patent/FR2645173B1/fr not_active Expired - Fee Related
• 1990
  • 1990-03-29 WO PCT/FR1990/000216 patent/WO1990011358A1/fr not_active Application Discontinuation
  • 1990-03-29 EP EP90907112A patent/EP0465602A1/de not_active Withdrawn
  • 1990-03-29 JP JP2506608A patent/JPH04506747A/ja active Pending
• 1991
  • 1991-09-27 OA OA60078A patent/OA09556A/fr unknown

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events