Nucleotide sequences derived from the genomes of the retroviruses HIV-1, HIV-2 and SIV, and their application to the amplification of these viral genomes and to the in vitro diagnosis of infections resulting from these viruses

The present invention relates to oligonucleotide sequences which can be used for the implementation of techniques for amplification of specific nucleic sequences of human immunodeficiency retrovirus of HIV type or of monkey immunodeficiency retrovirus of SIV type.

The invention relates in particular to the application of these sequences to diagnostic methods <u>in vitro</u> in humans, the infection of an individual with a retrovirus of the HIV type (currently HIV-1 and / or HIV-2).

The isolation and characterization of retroviruses grouped under the designations HIV-1 and HIV-2 have been described in European patent applications No. 85 / 905.513.9 and No. 87 / 400.151.4 respectively. These retroviruses have been isolated from several patients with symptoms of lymphadenopathy or Acquired Immunodeficiency Syndrome (AIDS).

HIV-2 type retroviruses, like HIV-1 type retroviruses, are characterized by a tropism for human T4 lymphocytes and by a cytopathogenic effect with regard to these lymphocytes when they multiply therein, thus causing between others generalized and persistent polyadenopathies, or AIDS.

Another retrovirus, called SIV-1, this name replacing the previously known name STLV-III, was isolated from the rhesus macaque monkey (MD DANIEL et al. Science, 228, 1201 (1985); NL LETWIN et al, Science, 230, 71 (1985) under the name "STLV-IIImac").

Another retrovirus, designated "STLV-III_AGM", (or SIV_AGM) was isolated from wild green monkeys. But unlike the viruses present in the rhesus macaque monkey, the presence of STLV-III_AGM does not seem to induce an AIDS-like disease in the African green monkey.

For the convenience of language, these viruses will no longer be designated in what follows only by the expression SIV (the expression SIV is the English abbreviation for "Simian Immunodeficency Virus" (Monkey immunodeficiency virus) possibly followed by an abbreviation designating the species of monkey from which they come, for example "MAC" for the macaque "or" AGM "for the African green monkey (abbreviation of" African Green Monkey ").

A strain of the SIV-1Mac retrovirus was deposited at the CNCM on February 7, 1986 under the number I-521.

Continued study of the HIV-1 and HIV-2 retroviruses has also led to the production of complementary DNA sequences (cDNAs) of the RNAs of their genome. The complete nucleotide sequence of a cDNA of a representative retrovirus of the HIV-2 class (HIV-2 ROD) was deposited on 02/21/1986 at the CNCM under the number I-522, under the reference name LAV-2 ROD.

Likewise, the complete nucleotide sequence of a cDNA of a representative retrovirus of the HIV-1 class is described by WAIN HOBSON, SONIGO, COLE, DANOS and ALIZON in CEll (January 1985).

Also for the convenience of language, viruses of the HIV-1 and HIV-2 type will sometimes be designated in what follows by the expression HIV.

Diagnostic methods <u>in vitro</u> infections with viruses of the HIV-1 or HIV-2 type currently existing, call for the detection of ANTI-HIV-1 or anti-HIV-2 antibodies possibly present in a biological sample (biopsy) or in a biological fluid , for example in a serum obtained, from the patient under study, by bringing this biological fluid into contact with extracts or antigens of HIV-1 or HIV-2, under conditions allowing the production of a possible immunological reaction of these extracts or antigens with these antibodies.

Such diagnostic methods risk being falsely negative, in particular in the case of a recent infection of an individual with HIV-type viruses.

Gene amplification techniques are a considerable supplement to the development of diagnostic methods <u>in vitro</u> particularly susceptible to viral diseases. Among these gene amplification techniques, mention may be made of the PCR (Polymerase Chain Reaction) technique as described in European patent applications No. 86 / 302.298.4 of 03/27/1986 and No. 87 / 300.203.4 of 01/09/1987, or even the technique called "Qβreplicase" described in Biotechnology, vol.6, page 1197 (October 1988) and that using a RNA polymerase (T7RNA polymerase) described in the patent application International No. WO89 / 01050. These techniques make it possible to improve the detection sensitivity of nucleic acids of viruses, and require the use of specific synthetic primers.

For the detection of HIV type viruses, the choice of primers is problematic. Indeed, due to the great variability of the nucleotide sequences of the viral genome, a primer conforming to the known sequence of a given isolate of a virus of the HIV type may fail to amplify certain viral variants of the HIV type. On the other hand, even if a primer is chosen in a conserved region of the genome of an HIV virus to another, its "good functioning" is not guaranteed and can give rise to poor amplification yields.

The present invention precisely provides oligonucleotide primers allowing, among other things, the amplification, in particular for diagnostic purposes, of the genome of all viruses of the HIV and SIV type, with yields considered to be maximum in the current state of the art and above all avoiding the presence of numerous non-specific bands.

The primers of the present invention are both specific for viruses of the HIV-1 group and / or viruses of the HIV-2 and SIV groups, and are insensitive to variations in the genome of these viruses.

The subject of the present invention is oligonucleotide primers, of around 15 to 30 nucleotides, which can be used for genomic amplification of viruses of the HIV-1 type and / or of the HIV-2 and SIV type.

The invention relates to any nucleotide sequence characterized in that its sequence:<ul id="ul0001" list-style="dash" compact="compact"><li>either is chosen from those which are contained in one of the nucleotide sequences included in the gag, vpr and pol genes of the HIV-1 Bru, HIV-1 Mal, HIV-1 Eli, HIV-2 ROD and SIV MAC viruses, or in the nef2, vif2 and vpx genes of the HIV-2 ROD, and SIV MAC viruses, or in the env, nef1, vif1 and vpr genes of the HIV-1 Bru, HIV-1 Mal, and HIV-1 Eli viruses, and more particularly among those contained in the nucleotide sequences defined below,</li><li>either (in particular for the longest sequences) contains one of the abovementioned nucleotide sequences derived from HIV-1 Bru, or HIV-1 Mal, or HIV-1 Eli or HIV-2 ROD or SIVMac, or contains a complementary nucleotide sequence of one of these latter sequences, it being understood that any additional nucleotides which "extend" over the nucleotide sequence of the genus in question, on the 3 ′ or 5 ′ end side, preferably coincide with those which are placed below the 5 ′ or 3 ′ ends corresponding within the complete sequence of the viruses of the HIV-1, HIV-2 or SIV MAC type, mentioned above,</li><li>either, if this nucleotide sequence is not identical to one of the above nucleotide sequences, or is not complementary to one of these sequences, is nevertheless capable of hybridizing with a nucleotide sequence derived from HIV- 1 Bru, HIV-1 Mal, HIV-1 Eli, and / or with a nucleotide sequence derived from the HIV-2 ROD or SIV MAC virus mentioned above. Hybridization can be carried out at a temperature of 60 ° C ± 1 ° C (preferably 60 ° C ± 0.5 ° C), recommended for optimum performance.</li></ul>

The numbering of the nucleotides mentioned below corresponds to that used in the reference manual "Human Retrovirus and AIDS-1989" published by the "Los Alamos National Laboratory- New Mexico - USA".

(The sequences of the HIV-1 Mal, HIV-1 Eli viruses have been described by MONTAGNIER, SONIGO, WAIN-HOBSON and ALIZON in European patent application No. 86.401380 of 06/23/86).

The sequences of the invention are synthesized on a synthesizer marketed by Applied Biosystems (phosphoro-amidites method, or on any other device using a similar method.

The invention relates more particularly to the oligonucleotide sequences characterized by the following nucleotide sequences (represented in the 5 ′ → 3 ′ direction; the initials “S” and “AS” indicate whether the oligonucleotide is sense or antisense, ie - say if the oligonucleotide is oriented respectively in the 5 '○ → 3' direction or in the 3 '○ → 5' direction):<ul id="ul0002" list-style="none"><li>1) sequences common to the genomes of the HIV-1, HIV-2 and SIV viruses (the series of figures spaced apart by a line indicate the position of the nucleotides on the genomes corresponding respectively to the HIV-1 Bru virus, HIV-1 Mal virus, HIV-1 Eli, HIV-2 ROD and SIV):<ul id="ul0003" list-style="none" compact="compact"><li>. specific sequences of the gag gene of the genome of the above-mentioned viruses (gene coding for a group of antigens specific to the nucleoid of these viruses). Certain variants can be made at certain positions of the nucleotide sequences indicated below, without the hybridization properties of these nucleotide sequences with the genes of viruses of the HIV and / or SIV type being affected. The nucleotide sequences comprising these variants are shown below the initial nucleotide sequences from which they are derived by replacement of one or more bases. The bases modified with respect to those of the initial nucleotide sequences are indicated in any letter vertically from the positions corresponding to the bases which have been replaced in these initial sequences; while the bases of the initial sequences which have not been replaced in the sequences comprising these variants are indicated using dotted lines. The synthesis of the primers is done using all the variants simultaneously. It is the mixture of all the variants for a given sequence which is used in the tests.<img file="EP0806484A2_D0001.tif" /></li><li>. specific sequences of the vpr gene<img file="EP0806484A2_D0002.tif" /><img file="EP0806484A2_D0003.tif" /></li><li>. specific pol gene sequences:</li></ul><img file="EP0806484A2_D0004.tif" /></li><li>2) sequences common to the genomes of the HIV-2 and SIV viruses (the series of figures spaced apart by a line indicate the position of the nucleotides on the genomes corresponding respectively to the HIV-2 ROD and SIV-MAC viruses).<ul id="ul0004" list-style="none"><li>. specific sequences of the nef2 gene (coding for a negative factor of 27 kD)<img file="EP0806484A2_D0005.tif" /></li><li>. specific sequences of the vif2 gene (coding for an infectivity factor of 23 kD)<img file="EP0806484A2_D0006.tif" /></li><li>. specific sequences of the vpx gene (coding for a 12 kD protein)</li></ul><img file="EP0806484A2_D0007.tif" /></li><li>3 °) Sequences common to the genomes of the HIV-1 Bru, HIV-1 Mal, and HIV-1 Eli viruses (the series of figures spaced apart by a line indicate the position of the nucleotides on the genomes corresponding respectively to the HIV-1 Bru viruses, HIV-1 MAl and HIV-1 Eli).<ul id="ul0005" list-style="none"><li>. specific env gene sequences (encoding envelope proteins)<img file="EP0806484A2_D0008.tif" /></li><li>. specific sequences of the nef1 gene<img file="EP0806484A2_D0009.tif" /><img file="EP0806484A2_D0010.tif" /></li><li>. specific gene 1 sequences<img file="EP0806484A2_D0011.tif" /></li><li>. specific sequences of the vpu gene</li></ul></li></ul><img file="EP0806484A2_D0012.tif" />

The subject of the invention is also the sequences (or primers) having a nucleotide structure complementary to those of the primers defined above.

It also relates to the nucleotide sequences exhibiting certain mutations with respect to those defined above without the hybridization properties, as defined above, of these sequences being modified. The percentage of nucleotides different from those constituting the sequences described above, without however affecting the hybridization properties of the sequences of the invention, can reach 40%.

In general, in the case of a primer (S), a larger number of mutations will be tolerated on the 5 'side than on the 3' side of the primer, the 3 'side in front of' hybridize perfectly with a specific strand of a nucleic sequence to allow the amplification of this sequence. In the case of an anti-sense primer (AS), tolerance is allowed on the 3 'side.

A subject of the invention is also the primers as defined above and comprising a conservation of at least 5 bases on each side, the middle part comprising modifications, without the above hybridization properties being modified.

One of the characteristics of the oligonucleotide primers of the invention is to give a clear amplification band, generally devoid of non-specific bands when the technical indications for use described in the present invention are used. This milk is due to the length of the primers which can reach 27 bases which increases the specificity of hybridization, as well as to the drastic conditions of use which make it possible to eliminate the parasitic associations. The specificity for each type of virus is a function, in addition to the percentage of homology with the reference matrix, of the length of the primers, which can reach, for an acceptable yield, up to 40 bases.

The invention also extends to primers as described above linked at the level of their 5 ′ end to a promoter for the implementation of a method of genomic amplification by synthesis of multiple copies of DNA or of RNA as described in European patent application n ° 88 / 307.102.9 dated 08/01/1988.

The subject of the invention is in particular the use of the primers described above for the implementation of a method for gene amplification of nucleic sequences of viruses of the HIV-1 and / or HIV-2 type, and / or SIV , this method being applicable to diagnosis <u>in vitro</u> potential infection of an individual with a virus of the HIV-1 and / or HIV-2 type or of an animal with at least one of the three viruses (HIV-1, HIV-2, SIV).

This diagnostic method <u>in vitro</u> of the invention is carried out from a biological sample (for example a biological fluid such as serum, circulating blood lymphocytes) obtained from a patient under study, and mainly comprises the following steps:<ul id="ul0006" list-style="dash" compact="compact"><li>a step of extracting the nucleic acid to be detected belonging to the genome of the virus of the HIV-1 and / or HIV-2 and / or SIV type possibly present in the abovementioned biological sample, and, where appropriate, a step of treatment using a reverse transcriptase of said nucleic acid if the latter is in the form of RNA in order to obtain a double-stranded nucleic acid (this last step being further designated below by step of reverse transcription of viral RNA),</li><li>a cycle comprising the following stages:<ul id="ul0007" list-style="none" compact="compact"><li>. denaturation of the double-stranded nucleic acid to be detected, which leads to the formation of a single-stranded nucleic acid,</li><li>. hybridization of each of the nucleic acid strands, obtained during the preceding denaturation step, with at least one primer according to the invention, by bringing the above-mentioned strands into contact with at least one pair of primers according to invention under the hybridization conditions defined below,</li><li>. formation from DNA primers complementary to the strands on which they are hybridized in the presence of a polymerization agent (DNA polymerase) and four different nucleoside triphosphate (dNTP), which leads to the formation of a larger number of double stranded nucleic acids to be detected in the previous denaturation step,</li></ul> this cycle being repeated a number of times determined to obtain said nucleic sequence to be detected, possibly present in the biological sample in a sufficient proportion to allow its detection,</li><li>a step of detecting the possible presence of the nucleic acid belonging to the genome of the virus of the HIV-1 and / or HIV-2 and / or SIV type in the biological sample.</li></ul>

The hybridization step described above is advantageously carried out at 60 ° C for 1 minute 30 seconds in the "10 X buffer" buffer whose composition (in final concentration of use) is indicated below.

The diagnostic method <u>in vitro</u> of the invention can be carried out either from viral RNA, or from complementary, episomal or integrated DNA.

In fact, the genomes of the HIV and SIV viruses are in the form of RNA or DNA depending on the location of the virus in the body.

When the virus is located inside the cells of the organism, especially inside the blood cells, its RNA is copied into DNA by a reverse transcriptase. On the other hand, the genome of HIV type viruses in an extracellular medium, especially in the blood, remains in the form of RNA.

The extraction step according to the invention of the viral DNA contained in the cells of the biological sample recommended by the inventors - in addition to the conventional phenol chloroform method - has the following steps:<ul id="ul0008" list-style="none" compact="compact"><li>. suspension of the cell pellet in 0.5 ml of pyrolized water in a Potter large piston,</li><li>. shredding of cells known as "round trip",</li><li>. addition of Triton X100 for 1 final concentration of 0.1%,</li><li>. heat denaturation for 15 to 25 minutes at 100 ° C,</li><li>. short centrifugation to remove only cellular debris,</li><li>. DNA precipitation overnight at -20 ° C by adding 2.5 volumes of absolute ethanol and 10% of the final volume of 3 Molar sodium acetate. The DNA is then recovered and then resuspended in pyrolyzed water after having been washed twice with ethanol at 70 °. It should be noted that this method allows the joint precipitation of DNA and RNA, which authorizes the detection of the genomic message of viruses of HIV or SIV types by using the method called "direct DNA PCR" or by that called "PCR -RNA ".</li></ul>

The viral RNA extraction step is generally carried out in a conventional manner known to those skilled in the art.

After extraction of the RNA, an additional step of transformation of the RNA, single strand into double stranded DNA is necessary to be carried out when the diagnosis <u>in vitro</u> of the invention is produced from biological samples containing viruses of the HIV-1 and / or HIV-2 and / or SIV type, the genomes of which are in the form of RNA.

This transformation of the RNA into DNA is carried out by treatment of the RNA obtained after extraction of the biological sample, in particular of the serum, in an appropriate medium using a reverse transcriptase.

The invention more particularly relates inter alia to a diagnostic method <u>in vitro</u> as defined above, in which the step of reverse transcription of the viral RNA is carried out in the following manner:<ul id="ul0009" list-style="dash" compact="compact"><li>10 μg of extracted RNA resuspended in water is brought into contact with the pair of primers at a concentration of 40 μM each, in a final volume of 40 μl. The whole is denatured at 100 ° C for 10 minutes then immersed in ice water,</li><li>10 μl of the following mixture are added: 5 μl of the “10 X buffer” buffer described below + 1 unit of reverse transcriptase (of AMV (Avian Myeloblastosis Virus) or of MuMLV (Moloney Leukemia Virus)) + 1 Tag polymerase unit + 1 µl of the mixture of 4 dNTPs at 25 mM each + QSP water 10 µl. The final volume is therefore 50 µl.</li></ul>

This reaction takes place in two stages:<ul id="ul0010" list-style="dash" compact="compact"><li>a) first step: manufacture of the cDNA by the action of reverse transcriptase at 42 ° C for 13 minutes,</li><li>b) 2nd step: conventional gene amplification: heating to 95 ° C. for 3 minutes to destroy the reverse transcriptase and allow the dehybridization / hybridization step, then the cycle described above for gene amplification is started.</li></ul>

The invention more particularly relates to a diagnostic method <u>in vitro</u> as described above, in which the denaturing step is carried out in the presence of the pair (s) of primers (or primers) of the invention. Indeed, as has been specified above, one of the characteristics of the oligonucleotides (or primers) of the invention is to give a clear amplification band, generally devoid of non-specific bands, when they are used under the conditions that follow :<ul id="ul0011" list-style="dash" compact="compact"><li><u>hybridization</u> : the primers (1 μl of a 40 μmolar solution (40 μM) of each primer) are placed in the presence of the template DNA (100 to 300 ng) for the first denaturation-reassociation step; the mixture is heated for 10 minutes at 100 ° C. and then the tubes containing this mixture of DNA-matrix and primers are immersed in water containing ice in order to increase the rate of DNA-matrix / primers reassociation. Primers should be used at a final concentration in the subsequent amplification step of 0.8 µM each.</li><li><u>amplification</u> : the 4 A dNTP is each added to the preceding medium, being used at 0.5 μmolar in final solution (50 μl), and one unit of Taq-polymerase for a reaction medium of 50 μl; this step is carried out in an amplification buffer of the present invention, generally designated under the name of "10 X buffer", the composition (when diluted to 1/10 °) is as follows: Tris-HCl, pH 8.9: 50 mM; (NH₄)₂ SO₄ : 15 mM; MgCl2: 5 mM; β-mercapto-ethanol: 10 mM; gelatin: 0.25 mg / ml. 5 μl of this buffer and water qs 50 μl are added to the preceding medium.</li></ul>

The amplification cycles are carried out as follows: 30 to 40 cycles composed of:<ul id="ul0012" list-style="none" compact="compact"><li>. 94 ° C for 10 seconds (denaturation),</li><li>. 60 ° C for 1 minute 30 (hybridization),</li><li>. 78 ° C for 1 minute 30 (elongation).</li></ul>

Everything will be followed by a single cycle at 78 ° C for 15 minutes.

The accuracy of the temperatures indicated to within ± 0.3 ° C, as well as their stability during the various cycles, represent essential conditions for obtaining maximum yields as well as the absence of non-specific bands.

The optimal DNA concentration is 100 to 300 ng for genomic DNA extracted from cells (from patients or in culture, from mammals or others).

It goes without saying that the above conditions represent optimal conditions for a final reaction medium of 50 μl, and that these conditions can be modified as a function of the final volume of the reaction medium.

The use of several pairs of different primers (or couple cocktails) of the invention allows either the cross-detection of several types of virus of the HIV and / or SIV type, or the simultaneous detection of several genes of the same virus HIV and / or SIV type.

By way of example of preferred pairs of primers which can be used in the context of the present invention, mention may be made of the following pairs of primers:<ul id="ul0013" list-style="dash" compact="compact"><li>MMy1-MMy4, MMy2-MMy4, MMy1-MMy3, MMy18-MMy19, MMy4bis-MMy28bis, MMy28-MMy29bis, MMy29-MMy30bis, MMy31-MMy32bis, especially for diagnosis <u>in vitro</u> of an individual's infection with HIV-1 and / or HIV-2</li><li>MMy5-MMy8, MMy6-MMy8, MMy7-MMy8, MMy5-MMy7bis, MMy6-MMy7bis, MMy9-MMy11, MMy10-My11, MMy9-MMy10bis, MMy26-MMy5bis, MMy8bis-MMy9bis, MMy8bis-MMy89 MMy17, MMy15-MMy16bis, MMy16-MMy17, MMy25-MMy27, MMy26-MMy27, especially for diagnosis <u>in vitro</u> infection of an individual with HIV-1,</li><li>MMy20-MMy22, MMy20-MMy21bis, MMy21-MMy22, MMy23-MMy24, MMy12-MMy14, MMy12-MMy13bis, for diagnosis <u>in vitro</u> of an individual's infection with HIV-2.</li></ul>

The polymerization agent used in the elongation stage of the cycle is a thermostable DNA polymerase, in particular Taq polymerase, the amplifiose from the firm Appligène or any thermostable DNA polymerase that can be marketed.

Generally speaking, the diagnostic method cycle <u>in vitro</u> of the invention is repeated between 30 and 40 times.

The diagnostic method <u>in vitro</u> of the invention also makes it possible, as a function of the pairs of nucleotide primers used, to selectively detect the genes of viruses of the HIV and / or SIV type present in the biological sample.

The pairs of primers which can be used, by way of examples, for the above-mentioned gene by gene diagnostic method of the invention, are the following:<ul id="ul0014" list-style="dash" compact="compact"><li>MMy1-MMy4, MMy2-MMy4, MMy1-MMy3, MMy4bis-MMy28bis for the gag gene,</li><li>MMy18-MMy19 for the vpr gene,</li><li>MMy5-MMy8, MMy6-MMy8, MMy7-MMy8, MMy5-MMy7bis, MMy6-MMy7bis, MMy26-MMy5bis, MMy8bis-MMy9bis, MMy8bis-MMy89, MMy89bis-MMy9bis for the env gene,</li><li>MMy9-MMy11, MMy9-MMy10bis, MMy10-MMy11 for the nef1 gene,</li><li>MMy15-MMy17, MMy15-MMy16bis, MMy16-MMy17, for the vif1 gene,</li><li>MMy20-MMy22, MMy20-MMy21bis, MMy21-MMy22 for quick 2,</li><li>MMy23-MMy24 for vpx,</li><li>MMy12-MMy14, MMy12-MMy13bis, MMy13-MMy14 for nef2,</li><li>MMy25-MMy27, MMY26-MMy27 for the vpu gene,</li><li>MMy28-MMy29bis, MMy29-MMy30bis, MMy30-MMy31bis, MMy31-MMy32bis for the pol gene.</li></ul>

However, the combinations between primers "S" and "AS" described above are not limiting and can be varied as desired by the user.

The sizes of the nucleotide fragments synthesized using the pairs of primers mentioned above as examples, are indicated in the following Tables I to XI: (the figures indicated in the tables below represent the number of nucleotides of the fragments synthesized, and the "dashes" indicate that the pairs of primers tested do not make it possible to characterize the corresponding viral strains). <tables id="tabl0001" num="0001"><table frame="all"><title>Table I</title><tgroup cols="5" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="31.50mm" /><colspec colnum="2" colname="col2" colwidth="31.50mm" /><colspec colnum="3" colname="col3" colwidth="31.50mm" /><colspec colnum="4" colname="col4" colwidth="31.50mm" /><colspec colnum="5" colname="col5" colwidth="31.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" rowsep="0" /><entry namest="col2" nameend="col3" align="center">gag</entry><entry namest="col4" nameend="col5" align="center">gag</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">MMy1-MMy3</entry><entry namest="col3" nameend="col3" align="center">MMy1-MMy4</entry><entry namest="col4" nameend="col4" align="center">MMy2-MMy4</entry><entry namest="col5" nameend="col5" align="center">MMy4bis-MMy28bis</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">HIV1-BRU</entry><entry namest="col2" nameend="col2" align="right">265</entry><entry namest="col3" nameend="col3" align="right">750</entry><entry namest="col4" nameend="col4" align="right">532</entry><entry namest="col5" nameend="col5" align="right">671</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-MAL</entry><entry namest="col2" nameend="col2" align="right">282</entry><entry namest="col3" nameend="col3" align="right">785</entry><entry namest="col4" nameend="col4" align="right">556</entry><entry namest="col5" nameend="col5" align="right">671</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-ELI</entry><entry namest="col2" nameend="col2" align="right">265</entry><entry namest="col3" nameend="col3" align="right">750</entry><entry namest="col4" nameend="col4" align="right">538</entry><entry namest="col5" nameend="col5" align="right">674</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV2-ROD</entry><entry namest="col2" nameend="col2" align="right">354</entry><entry namest="col3" nameend="col3" align="right">845</entry><entry namest="col4" nameend="col4" align="right">544</entry><entry namest="col5" nameend="col5" align="right">663</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">IF V</entry><entry namest="col2" nameend="col2" align="right">343</entry><entry namest="col3" nameend="col3" align="right">844</entry><entry namest="col4" nameend="col4" align="right">544</entry><entry namest="col5" nameend="col5" align="right">668</entry></row></tbody></tgroup></table></tables><tables id="tabl0002" num="0002"><table frame="all"><title>Table II</title><tgroup cols="5" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="31.50mm" /><colspec colnum="2" colname="col2" colwidth="31.50mm" /><colspec colnum="3" colname="col3" colwidth="31.50mm" /><colspec colnum="4" colname="col4" colwidth="31.50mm" /><colspec colnum="5" colname="col5" colwidth="31.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" rowsep="0" /><entry namest="col2" nameend="col3" align="center">approx</entry><entry namest="col4" nameend="col5" align="center">approx</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">MMy5-MMy7bis</entry><entry namest="col3" nameend="col3" align="center">MMy5-MMy8</entry><entry namest="col4" nameend="col4" align="center">MMy6-MMy7bis</entry><entry namest="col5" nameend="col5" align="center">MMy6-MMy8</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">HIV1-BRU</entry><entry namest="col2" nameend="col2" align="right">480</entry><entry namest="col3" nameend="col3" align="center">953</entry><entry namest="col4" nameend="col4" align="center">330</entry><entry namest="col5" nameend="col5" align="center">803</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-MAL</entry><entry namest="col2" nameend="col2" align="right">471</entry><entry namest="col3" nameend="col3" align="center">944</entry><entry namest="col4" nameend="col4" align="center">321</entry><entry namest="col5" nameend="col5" align="center">794</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-ELI</entry><entry namest="col2" nameend="col2" align="right">471</entry><entry namest="col3" nameend="col3" align="center">941</entry><entry namest="col4" nameend="col4" align="center">321</entry><entry namest="col5" nameend="col5" align="center">791</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV2-ROD</entry><entry namest="col2" nameend="col2" align="right">-</entry><entry namest="col3" nameend="col3" align="center">-</entry><entry namest="col4" nameend="col4" align="center">-</entry><entry namest="col5" nameend="col5" align="center">-</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">IF V</entry><entry namest="col2" nameend="col2" align="right">-</entry><entry namest="col3" nameend="col3" align="center">-</entry><entry namest="col4" nameend="col4" align="center">-</entry><entry namest="col5" nameend="col5" align="center">-</entry></row></tbody></tgroup></table></tables><tables id="tabl0003" num="0003"><table frame="all"><title>Table III</title><tgroup cols="4" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" rowsep="0" /><entry namest="col2" nameend="col3" align="center">approx</entry><entry namest="col4" nameend="col4" align="center">approx</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">MMy7-MMy8</entry><entry namest="col3" nameend="col3" align="center">MMy26-MMy5bis</entry><entry namest="col4" nameend="col4" align="center">MMy8bis-MMy9bis</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">HIV1-BRU</entry><entry namest="col2" nameend="col2" align="right">498</entry><entry namest="col3" nameend="col3" align="right">691</entry><entry namest="col4" nameend="col4" align="right">1038</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-MAL</entry><entry namest="col2" nameend="col2" align="right">498</entry><entry namest="col3" nameend="col3" align="right">691</entry><entry namest="col4" nameend="col4" align="right">1041</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-ELI</entry><entry namest="col2" nameend="col2" align="right">495</entry><entry namest="col3" nameend="col3" align="right">679</entry><entry namest="col4" nameend="col4" align="right">1038</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV2-ROD</entry><entry namest="col2" nameend="col2" align="right">-</entry><entry namest="col3" nameend="col3" align="right">-</entry><entry namest="col4" nameend="col4" align="right">-</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">IF V</entry><entry namest="col2" nameend="col2" align="right">-</entry><entry namest="col3" nameend="col3" align="right">-</entry><entry namest="col4" nameend="col4" align="right">-</entry></row></tbody></tgroup></table></tables><tables id="tabl0004" num="0004"><table frame="all"><title>Table IV</title><tgroup cols="3" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" rowsep="0" /><entry namest="col2" nameend="col2" align="center">approx</entry><entry namest="col3" nameend="col3" align="center">approx</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">MMy8bis-MMy89</entry><entry namest="col3" nameend="col3" align="center">MMy89bis-MMy9bis</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">HIV1-BRU</entry><entry namest="col2" nameend="col2" align="center">411</entry><entry namest="col3" nameend="col3" align="center">646</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-MAL</entry><entry namest="col2" nameend="col2" align="center">411 :</entry><entry namest="col3" nameend="col3" align="center">649</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-ELI</entry><entry namest="col2" nameend="col2" align="center">411</entry><entry namest="col3" nameend="col3" align="center">646</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV2-ROD</entry><entry namest="col2" nameend="col2" align="center">-</entry><entry namest="col3" nameend="col3" align="center">-</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">IF V</entry><entry namest="col2" nameend="col2" align="center">-</entry><entry namest="col3" nameend="col3" align="center">-</entry></row></tbody></tgroup></table></tables><tables id="tabl0005" num="0005"><table frame="all"><title>Table V</title><tgroup cols="4" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" rowsep="0" /><entry namest="col2" nameend="col2" align="center">nef1</entry><entry namest="col3" nameend="col4" align="center">nef1</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">MMy9-MMy10bis</entry><entry namest="col3" nameend="col3" align="center">MMy9-MMy11</entry><entry namest="col4" nameend="col4" align="center">MMy10-MMy11</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">HIV1-BRU</entry><entry namest="col2" nameend="col2" align="left">293</entry><entry namest="col3" nameend="col3" align="center">660</entry><entry namest="col4" nameend="col4" align="center">388</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-MAL</entry><entry namest="col2" nameend="col2" align="left">302</entry><entry namest="col3" nameend="col3" align="center">660</entry><entry namest="col4" nameend="col4" align="center">388</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-ELI</entry><entry namest="col2" nameend="col2" align="left">296</entry><entry namest="col3" nameend="col3" align="center">663</entry><entry namest="col4" nameend="col4" align="center">388</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV2-ROD</entry><entry namest="col2" nameend="col2" align="left">-</entry><entry namest="col3" nameend="col3" align="center">-</entry><entry namest="col4" nameend="col4" align="center">-</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">IF V</entry><entry namest="col2" nameend="col2" align="left">-</entry><entry namest="col3" nameend="col3" align="center">-</entry><entry namest="col4" nameend="col4" align="center">-</entry></row></tbody></tgroup></table></tables><tables id="tabl0006" num="0006"><table frame="all"><title>Table VI</title><tgroup cols="4" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" rowsep="0" /><entry namest="col2" nameend="col3" align="center">nef2</entry><entry namest="col4" nameend="col4" align="center">nef2</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">MMy12-MMy13bis</entry><entry namest="col3" nameend="col3" align="center">MMy12-MMy14</entry><entry namest="col4" nameend="col4" align="center">MMy13-MMy14</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">HIV1-BRU</entry><entry namest="col2" nameend="col2" align="right">-</entry><entry namest="col3" nameend="col3" align="left">-</entry><entry namest="col4" nameend="col4" align="left">-</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-MAL</entry><entry namest="col2" nameend="col2" align="right">-</entry><entry namest="col3" nameend="col3" align="left">-</entry><entry namest="col4" nameend="col4" align="left">-</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-ELI</entry><entry namest="col2" nameend="col2" align="right">-</entry><entry namest="col3" nameend="col3" align="left">-</entry><entry namest="col4" nameend="col4" align="left">-</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV2-ROD</entry><entry namest="col2" nameend="col2" align="right">400</entry><entry namest="col3" nameend="col3" align="left">792</entry><entry namest="col4" nameend="col4" align="left">415</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">IF V</entry><entry namest="col2" nameend="col2" align="right">400</entry><entry namest="col3" nameend="col3" align="left">755</entry><entry namest="col4" nameend="col4" align="left">378</entry></row></tbody></tgroup></table></tables><tables id="tabl0007" num="0007"><table frame="all"><title>Table VII</title><tgroup cols="4" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" rowsep="0" /><entry namest="col2" nameend="col3" align="center">vif1</entry><entry namest="col4" nameend="col4" align="center">vif1</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">MMy15-MMy16bis</entry><entry namest="col3" nameend="col3" align="center">MMy15-MMy17</entry><entry namest="col4" nameend="col4" align="center">MMy16-MMy17</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">HIV1-BRU</entry><entry namest="col2" nameend="col2" align="center">333</entry><entry namest="col3" nameend="col3" align="center">603</entry><entry namest="col4" nameend="col4" align="center">293</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-MAL</entry><entry namest="col2" nameend="col2" align="center">333</entry><entry namest="col3" nameend="col3" align="center">603</entry><entry namest="col4" nameend="col4" align="center">293</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-ELI</entry><entry namest="col2" nameend="col2" align="center">333</entry><entry namest="col3" nameend="col3" align="center">603</entry><entry namest="col4" nameend="col4" align="center">293</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV2-ROD</entry><entry namest="col2" nameend="col2" align="center">-</entry><entry namest="col3" nameend="col3" align="center">-</entry><entry namest="col4" nameend="col4" align="center">-</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">IF V</entry><entry namest="col2" nameend="col2" align="center">-</entry><entry namest="col3" nameend="col3" align="center">-</entry><entry namest="col4" nameend="col4" align="center">-</entry></row></tbody></tgroup></table></tables><tables id="tabl0008" num="0008"><table frame="all"><title>Table VIII</title><tgroup cols="4" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" rowsep="0" /><entry namest="col2" nameend="col2" align="center">vpr</entry><entry namest="col3" nameend="col4" align="center">lively2</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">MMy18-MMy19</entry><entry namest="col3" nameend="col3" align="center">MMy20-MMy21bis</entry><entry namest="col4" nameend="col4" align="center">MMy20-MMy22</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">HIV1-BRU</entry><entry namest="col2" nameend="col2" align="right">281</entry><entry namest="col3" nameend="col3" align="right">-</entry><entry namest="col4" nameend="col4" align="right">-</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-MAL</entry><entry namest="col2" nameend="col2" align="right">281</entry><entry namest="col3" nameend="col3" align="right">-</entry><entry namest="col4" nameend="col4" align="right">-</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-ELI</entry><entry namest="col2" nameend="col2" align="right">281</entry><entry namest="col3" nameend="col3" align="right">-</entry><entry namest="col4" nameend="col4" align="right">-</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV2-ROD</entry><entry namest="col2" nameend="col2" align="right">319</entry><entry namest="col3" nameend="col3" align="right">352</entry><entry namest="col4" nameend="col4" align="right">659</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">IF V</entry><entry namest="col2" nameend="col2" align="right">308</entry><entry namest="col3" nameend="col3" align="right">352</entry><entry namest="col4" nameend="col4" align="right">656</entry></row></tbody></tgroup></table></tables><tables id="tabl0009" num="0009"><table frame="all"><title>Table IX</title><tgroup cols="3" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" rowsep="0" /><entry namest="col2" nameend="col2" align="center">lively2</entry><entry namest="col3" nameend="col3" align="center">vpx</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">MMy21-MMy22</entry><entry namest="col3" nameend="col3" align="center">MMy23-MMy24</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">HIV1-BRU</entry><entry namest="col2" nameend="col2" align="right">-</entry><entry namest="col3" nameend="col3" align="center">-</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-MAL</entry><entry namest="col2" nameend="col2" align="right">-</entry><entry namest="col3" nameend="col3" align="center">-</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-ELI</entry><entry namest="col2" nameend="col2" align="right">-</entry><entry namest="col3" nameend="col3" align="center">-</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV2-ROD</entry><entry namest="col2" nameend="col2" align="right">329</entry><entry namest="col3" nameend="col3" align="center">329</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">IF V</entry><entry namest="col2" nameend="col2" align="right">326</entry><entry namest="col3" nameend="col3" align="center">329</entry></row></tbody></tgroup></table></tables><tables id="tabl0010" num="0010"><table frame="all"><title>Paintings</title><tgroup cols="4" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" rowsep="0" /><entry namest="col2" nameend="col3" align="center">vpu</entry><entry namest="col4" nameend="col4" align="center">pol</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">MMy25-MMy27</entry><entry namest="col3" nameend="col3" align="center">MMy26-MMy27</entry><entry namest="col4" nameend="col4" align="center">MMy28-MMy29bis</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">HIV1-BRU</entry><entry namest="col2" nameend="col2" align="right">263</entry><entry namest="col3" nameend="col3" align="center">104</entry><entry namest="col4" nameend="col4" align="right">623</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-MAL</entry><entry namest="col2" nameend="col2" align="right">263</entry><entry namest="col3" nameend="col3" align="center">101</entry><entry namest="col4" nameend="col4" align="right">584</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-ELI</entry><entry namest="col2" nameend="col2" align="right">263</entry><entry namest="col3" nameend="col3" align="center">101</entry><entry namest="col4" nameend="col4" align="right">584</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV2-ROD</entry><entry namest="col2" nameend="col2" align="right">-</entry><entry namest="col3" nameend="col3" align="center">-</entry><entry namest="col4" nameend="col4" align="right">666</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">IF V</entry><entry namest="col2" nameend="col2" align="right">-</entry><entry namest="col3" nameend="col3" align="center">-</entry><entry namest="col4" nameend="col4" align="right">712</entry></row></tbody></tgroup></table></tables><tables id="tabl0011" num="0011"><table frame="all"><title>Table XI</title><tgroup cols="4" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" rowsep="0" /><entry namest="col2" nameend="col3" align="center">pol</entry><entry namest="col4" nameend="col4" align="center">pol</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">MMy29-MMy30bis</entry><entry namest="col3" nameend="col3" align="center">MMy30-MMy31bis</entry><entry namest="col4" nameend="col4" align="center">MMy31-MMy32bis</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">HIV1-BRU</entry><entry namest="col2" nameend="col2" align="right">742</entry><entry namest="col3" nameend="col3" align="right">869</entry><entry namest="col4" nameend="col4" align="right">826</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-MAL</entry><entry namest="col2" nameend="col2" align="right">742</entry><entry namest="col3" nameend="col3" align="right">869</entry><entry namest="col4" nameend="col4" align="right">826</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV1-ELI</entry><entry namest="col2" nameend="col2" align="right">742</entry><entry namest="col3" nameend="col3" align="right">869</entry><entry namest="col4" nameend="col4" align="right">826</entry></row><row><entry namest="col1" nameend="col1" align="left">HIV2-ROD</entry><entry namest="col2" nameend="col2" align="right">742</entry><entry namest="col3" nameend="col3" align="right">866</entry><entry namest="col4" nameend="col4" align="right">826</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">IF V</entry><entry namest="col2" nameend="col2" align="right">742</entry><entry namest="col3" nameend="col3" align="right">866</entry><entry namest="col4" nameend="col4" align="right">826</entry></row></tbody></tgroup></table></tables>

It should be noted that thanks to their arrangement on the genome, the primers used for amplification can be combined in such a way that they can be used as a probe, either after labeling with ³²p by kination, ie for the use in the technique of cold probes to verify the specificity of the amplification band observed during an analysis by "Southern transfer". In addition to the classic combination of primers so that a third oligonucleotide can serve as a specific internal probe, it should be noted the specific case of genes<u>vif1 / vpr</u> and <u>vif2 / vpx</u> due to the overlap of these genes, which allows cross-detection. In addition, during an analysis by sequencing of the amplified DNA, these oligonucleotides can be used as specific primer for DNA polymerase allowing a double sequencing in each direction, therefore a double reading of the sequences, thus removing any ambiguities d 'interpretation.

A subject of the invention is also the primers, as defined above, labeled, in particular in a radioactive or enzymatic manner, as well as their use as nucleotide probes, in particular within the framework of the diagnostic method. <u>in vitro</u> as described above.

The invention also relates to oligonucleotides as described above and comprising sugars in α conformation. Such oligonucleotides have the characteristic of reversing the direction of the dou ble helix formed with the matrix (strand of the virus genome), this double helix thus passing from the "S" state to the "AS" state.

The invention also relates to the oligonucleotides described above, certain nucleotides of which are methylated and / or contain one or more sulfur atoms, in particular on the adenines. Such oligonucleotides have the characteristic of increasing the stability of the double helix, and consequently of better hybridizing with the DNA strand to be amplified.

The invention also relates to the oligonucleotides as described above and which are in the form known as "modified bases" comprising nucleotides on which are covalently grafted chromophoric agents (flat aromatic molecules such as acridine orange), in particular according to the method described in the article by C. Hélène published in "la Vie des Sciences", reports, general series, volume 4, n ° 1, p. 17-37. Such oligonucleotides have the characteristic of being easily detectable , in particular by fluorescence.

The oligonucleotides of the invention can also be used for the implementation of a diagnostic method <u>in vitro</u> infection of monkeys (macaque, mangabeys monkey or green monkey) by the virus of the SIV type, this method incorporating the main characteristics of that described above.

The subject of the invention is also diagnostic kits for implementing diagnostic methods <u>in vitro</u> mentioned above. By way of example, a diagnostic kit of the present invention comprises:<ul id="ul0015" list-style="dash" compact="compact"><li>at least one pair of oligonucleotide primers according to the invention, each pair comprising a primer hybridizing to one of the strands of the nucleic acid sequence to be detected, and a primer hybridizing with the complementary strand of the latter under the conditions defined above,</li><li>reagents suitable for carrying out the amplification cycle, in particular DNA polymerase, and four different nucleotide triphosphate, and the reaction medium called "10 X buffer" described above.</li><li>one (or more) probe capable of being labeled, in particular by radioactivity or by the technique of cold probes, capable of specifically hybridizing with the amplified nucleic acid sequence (s) to be detected.</li></ul>

The invention also relates to the use of the primers of the invention indicated above for the implementation of a process for the synthesis of proteins encoded by the nucleotide sequences amplified using these primers.

By way of illustration, this method of protein synthesis comprises the amplification of nucleotide sequences of the genomes of viruses of the HIV or SIV type (coding for a specific protein and which has undergone, if necessary, certain modifications of their nucleotides) by contacting said sequences with at least one pair of primers according to the invention under the conditions described above, followed by the translation of these sequences thus amplified into proteins; this last step is carried out in particular by transformation of appropriate host cells using vectors containing said amplified sequences, and recovery of the proteins produced in these host cells.

The invention also relates to the polypeptides resulting from the translation of the nucleotide sequences (or primers) of the invention.

A subject of the invention is also the use of the antisense oligonucleotide primers as antiviral agents in general, in particular in the fight against AIDS, as well as pharmaceutical compositions containing these antisense primers in association with a pharmaceutically carrier. acceptable.

The invention also relates to the immunogenic compositions comprising one or more translation products of the nucleotide sequences according to the invention, and / or one or more translation products of the nucleotide sequences amplified according to the methods described above from the primers defined according to l invention, these translation products being associated with a pharmaceutically acceptable vehicle.

The invention relates to antibodies directed against one or more of the translation products described above (or in other words, capable of forming an immunological reaction with one or more translation products of the nucleotide sequences according to the invention, or one or more products of translation of the nucleotide sequences amplified from the primers defined according to the invention) and their use for the implementation of diagnostic methods <u>in vitro</u> infection of an individual with a virus of the HIV-1 and / or HIV-2 type, or of an animal with at least one of the three viruses (HIV-1, HIV-2, SIV) according to the processes known to those skilled in the art.

As an illustration, such a diagnostic method <u>in vitro</u> according to the invention comprises bringing a biological sample (in particular serum) taken from a patient under study, into contact with antibodies according to the invention, and detection using any suitable method (in particular using using labeled anti-immunoglobulins) of the immunological complexes formed between the antigens of the HIV or SIV type viruses possibly present in the biological sample and the said antibodies.

The invention also relates to diagnostic kits <u>in vitro</u> comprising antibodies according to the invention and, where appropriate, reagents suitable for demonstrating the immunological reaction formed between said antibodies and the antigens of the HIV or SIV viruses.

The invention also relates to a process for the preparation of the above-mentioned polypeptides, in particular those corresponding according to the universal genetic code to the nucleotide sequences (or primers) described above, this process being characterized in that, preferably starting from the C-terminal amino acid, the successive aminoacyles are successively condensed two by two in the required order, or aminoacyls and fragments previously formed and already containing several aminoacyl residues in the appropriate order, or several fragments previously thus prepared, it being understood that care has been taken beforehand to protect all the reactive functions carried by these aminoacyles or fragment with the exception of the amine functions of one and the carboxyl of the other or vice versa, which must normally intervene in the formation of peptide bonds, in particular after activation of the carboxyl function, according to the methods known in the synthesis of peptides and so on, step by step, up to the N-terminal amino acid.

For example, we will use the peptide synthesis technique in homogeneous solution described by Houbenweyl in "Meuthode der Organischen Chemie" (Method of Organic Chemistry) edited by E. Wunsch, vol. 15-I and II, THIEME, STUTTGART, 1974, or that of peptide synthesis in solid phase described by RD Merrifield in "Solid Phase Peptide Synthesis" (J. AM. CHEM. SOC.,<u>45</u>, 2149-2154).

The invention also relates to a process for preparing the nucleotide sequences (or primers) described above, this process comprising the following steps:<ul id="ul0016" list-style="dash" compact="compact"><li>incubation of the genomic DNA, isolated from one of the viruses of the HIV or SIV type mentioned above, with DNAase I, then addition of EDTA and purification by extraction with the phenol / chloroform / isoamyl alcohol mixture (25 / 24/1) then with ether,</li><li>processing of the DNA thus extracted with<u>Eco</u> R1 methylase in the presence of DTT, and purification by extraction as described above,</li><li>incubation of the DNA thus purified with the 4 deoxynucleotide triphosphates dATP, dCTP, dGTP, and dTTP in the presence of T4 DNA polymerase and DNA ligase <u>E. coli</u>, then purification according to the method described above,</li><li>the cloning of the nucleic acids thus obtained in an appropriate vector and the recovery of the nucleic acid sought using a suitable probe.</li></ul>

A particularly advantageous process for preparing the nucleotide sequences of the invention comprises the following steps:<ul id="ul0017" list-style="dash" compact="compact"><li>DNA synthesis using the automated β-cyanethyl phosphoramidite method described in Bioorganic Chemistry 4; 274-325 (1986),</li><li>cloning the nucleic acids thus obtained into an appropriate vector and recovering the nucleic acid by hybridization with an appropriate probe.</li></ul>

Another method for preparing the nucleotide sequences of the invention comprises the following steps:<ul id="ul0018" list-style="dash" compact="compact"><li>the assembly of chemically synthesized oligonucleotides, provided at their ends with different restriction sites, the sequences of which are compatible with the amino acid chain of the natural polypeptide according to the principle described in Proc. Natl. Acad. Sci. USA, 80; 7461-7465, (1983),</li><li>cloning the nucleic acids thus obtained into an appropriate vector and recovering the desired nucleic acid by hybridization with an appropriate probe.</li></ul>