Patent Application: US-20099098-A

Abstract:
composition comprising two pathogenic and / or infective agents associated with multiple sclerosis , namely a first agent which consists of a human virus possessing reverse transcriptase activity and related to a family of endogenous retroviral elements , or a variant of said virus , and a second agent , or a variant of said second agent , these two pathogenic and / or infective agents originating from the same viral strain chosen from the strains designated , respectively , pol - 2 deposited with the ecacc on jul . 22 , 1992 under accession number v92072202 and ms7pg deposited with the ecacc on jan . 8 , 1993 under accession number v93010816 , and from their variant strains .

Description:
obtaining msrv - 2 clones designated msrv - 2a , by amplification of the conserved regions of the genes for rna - dependent dna polymerases on a preparation of infective agent purified from lm7 line cell culture the molecular approach consisted in using a pcr technique ( 33 ) which makes it possible to amplify a relatively conserved region of the pol gene of exogenous and endogenous retroviruses , but also of viruses coding for an enzyme having reverse transcriptase ( rt ) activity , such as , in particular , the hepatatis b virus , and , implicitly , of any gene for rna - dependent dna polymerase or for an enzyme , displaying sufficient sequence homologies in the regions defined by the amplification primers used . this pcr technique was used on the nucleic acids extracted from a purified preparation of infective agent , obtained according to the protocol ( 34 ) from supernatants of the original lm7 culture ( 24 ) which were kept frozen at − 80 ° c . since that time . the fractions containing the peak of lm7 - like rt activity are taken up in one volume of a buffer containing guanidine thiocyanate ( 35 ), and are stored at − 80 ° c . until the nucleic acids are extracted according to the technique described by p . chomzynski ( 35 ). prior to the pcr reaction , the rna of the sample was transcribed into complementary dna ( cdna ) with so - called “ random ” primers ( mixed hexanucleotides ) using the “ cdna synthesis system plus ” kit ( amersham ) according to the manufacturer &# 39 ; s instructions , and on the basis of an approximate value , to the nearest log factor , of the amount of rna present in the sample . the dna obtained after pcr amplification of the cdna was inserted into a plasmid using the ta cloning ® kit ( british biotechnology ). the 2 μl of dna solution were mixed with 5 μl of sterile distilled water , 1 μl of a 10 - fold concentrated ligation buffer “ 10 × ligation buffer ”, 2 μl of “ pcr ™ vector ” ( 25 ng / ml ) and 1 μl of “ ta dna ligase ”. this mixture was incubated overnight at 12 ° c . the following steps were carried out in accordance with the instructions of the ta cloning ® kit . at the end of the procedure , the white colonies of recombinant bacteria were picked out in order to be cultured and to permit extraction of the plasmids incorporated according to the so - called “ miniprep ” procedure ( 36 ). the plasmid preparation from each recombinant colony was cut with a suitable restriction enzyme and analyzed on agarose gel . plasmids possessing an insert detected under uv light after staining the gel with ethidium bromide were selected for sequencing of the insert , after hybridization with a primer complementary to the sp6 promoter present on the cloning plasmid of the “ ta cloning kit ”. the reaction prior to sequencing was then performed according to the method recommended for the use of the sequencing kit “ prism ready reaction kit dye deoxyterminator cycle sequencing kit ” ( applied biosystems , ref . 401384 ), and automatic sequencing was carried out with an applied biosystems model 373 a “ automatic sequencer ” apparatus according to the manufacturer &# 39 ; s instructions . the sequences obtained were then analyzed using the mac vector ® and geneworks ® software on genebank ® computerized data bank for the nucleic acid sequences , and swiss prot ® for the amino acid sequences deduced from the reading frames revealed in the nucleic acid sequences . analysis of the sequences obtained from the viral sample originating from the thawed lm7 supernatants , and which was purified at the peak of reverse transcriptase activity on a sucrose gradient , revealed a majority population of clones ( approximately 42 % of the clones ), relative to the extent of individual representation of the other sequences ( always less than 5 %, or 10 % in a small number of cases ), displaying partial homologies with known retroviruses in the expected “ pol ” region . this clone is designated msrv2 - a and identified by seq id no10 ( see fig1 ). the region amplified between the pcr primers is homologous with the corresponding sequence msrv2 - b identified by seq id no11 ( see fig5 ), described in example 2 . the differences observed in the sequences located at the pcr primers is explained by the use of degenerate primers in mixture form , used under different technical conditions . interrogation of the genebank ® data bank , fully updated , did not enable an identical sequence or one displaying significant homologies to be revealed . this sequence is presented in fig1 . it possesses an open reading frame in frame with the two pcr primers to be found at the ends , but it is shorter than the set of known retroviral sequences in the expected region between these primers . a deletion of 45 base pairs ( 15 amino acids ) is observed therein following the sequence of the upstream primer , whereas the sequences preceding the downstream primer are present . however , the reading frame is open and uninterrupted over the whole of the sequence including the primers , and the deduced amino acid sequence displays a significant homology with the corresponding region of the known retroviruses . in the sequence lying inside the pcr primers , the amino acids glu , arg , gln , pro and asp , normally fairly well conserved in this pol region of retroviruses and of known viruses with reverse transcriptase activity ( 33 ), are to be found conserved at the correct positions in the reading frame of the novel sequence . lastly , in view of the fact that this sequence is sufficiently divergent from the retroviral sequences already described in the data banks , it may be suggested that the sequence in question belongs to a new infective and / or pathogenic agent , designated msrv - 2a . this agent is , in principle , on the basis of the analysis of the sequences obtained , related to a retrovirus but , in view of the technique used for obtaining this sequence , it may also be an rna virus whose genome codes for an enzyme which incidentally possesses reverse transcriptase activity , as is the case , for example , with the hepatitis b virus , hbv ( 33 ). furthermore , the random nature of the degenerate primers used for this pcr amplification technique may very well have permitted , as a result of unforeseen sequence homologies or of conserved sites in the gene for a related enzyme , the amplification of a nucleic acid originating from a prokaryotic or eukaryotic pathogenic and / or coinfective agent ( protist ). obtaining cones designated msrv - 1b and msrv - 2b , defining , respectively , a retrovirus msrv - 1 and a coinfective agent msrv2 , by “ nested ” pcr amplification of the conserved pol regions of retroviruses on virion preparations originating from the lm7pc and pli - 2 lines a pcr technique derived from the technique published by shih ( 33 ) was used . this technique enables all trace of contaminant dna to be removed by treating all the components of the reaction medium with dnase . it concomitantly makes it possible , by the use of different but overlapping primers in two successive series of pcr amplification cycles , to increase the chances of amplifying a cdna synthesized from an amount of rna which is small at the outset and further reduced in the sample by the spurious action of the dnase on the rna . in effect , the dnase is used under conditions of activity in excess which enable all trace of contaminant dna to be removed before inactivation of this enzyme remaining in the sample by heating to 85 ° c . for 10 minutes , this variant of the pcr technique described by shih ( 33 ) was used on a cdna synthesized from the nucleic acids of fractions of infective particles purified on a sucrose gradient according to the technique described by h . perron ( 34 ) from the “ pol - 2 ” isolate ( ecacc no . v92072202 ) produced by the pli - 2 line ( ecacc no . 92072201 ) on the one hand , and from the ms7pg isolate ( ecacc no . v93010816 ) produced by the lm7pc line ( ecacc no . 93010817 ) on the other hand . these cultures were obtained according to the methods which formed the subject of the patent applications published under nos wo 93 / 20188 and wo 93 / 20189 . after cloning the products amplified by this technique with the ta cloning kit ® and analysis of the sequence using the automatic sequencer as has been described in example 1 , the sequences were analyzed using the geneworks ® software on the latest available version of the genebank ® data bank . the sequences cloned and sequenced from these samples correspond , in particular , to two types of sequence : a first type of sequence , to be found in the majority of the clones ( 55 % of the clones originating from the pol - 2 isolates of the pli - 2 culture , and 67 % of the clones originating from the ms7pg isolates of the lm7pc cultures ), which corresponds to a family of “ pol ” sequences closely similar to , but different from , the endogenous human retrovirus designated erv - 9 or hserv - 9 , and a second type of sequence which corresponds to sequences very strongly homologous with the sequence attributed to an infective and / or pathogenic agent previously designated msrv - 2 . the first type of sequence , representing the majority of the clones , consists of sequences whose variability enables four subfamilies of sequences to be defined . these subfamilies are sufficiently similar to one another for it to be possible to consider them to be quasi - species originating from the same retrovirus , as is well known for the hiv - 1 retrovirus ( 37 ), or to be the outcome of interference with several endogenous proviruses coregulated in the producing cells . these more or less defective endogenous elements are sensitive to the same regulatory signals possibly generated by a replicative provirus , since they belong to the same family of endogenous retroviruses ( 38 ). this new family of endogenous retroviruses , or alternatively this new retroviral species from which the generation of quasi - species has been obtained in culture , and which contains a consensus of the sequences described below , is designated msrv - 1b . fig2 presents the general consensus sequences of the sequences of the different msrv - 1b clones sequenced in this experiment , these sequences being identified , respectively , by seq id no3 , seq id no4 , seq id no5 and seq id no6 . these sequences display a homology with respect to nucleic acids ranging from 70 % to 88 % with the hserv9 sequence referenced x57147 and m37638 in the genebank ® data base . the phylogenetic tree of these sequences is presented in fig3 . in this figure , the subfamilies a , b , c and d represent the sequences which have turned up preponderantly in similar experiments repeated subsequently , in the samples of pure rna of virions purified from the ms7pg and pol - 2 isolates . from these families of sequences , four “ consensus ” nucleic acid sequences representative of different quasi - species of a possibly exogenous retrovirus msrv - 1b , or of different subfamilies of an endogenous retrovirus msrv - 1b , have been defined . these representative consensus sequences are presented in fig4 with the translation into amino acids . a functional reading frame exists for each subfamily of these msrv - 1b sequences , and it can be seen that the functional open reading frame corresponds in each instance to the amino acid sequence appearing on the second line under the nucleic acid sequence . the general consensus of the msrv - 1b sequence , identified by seq id no7 and obtained by this pcr technique in the “ pol ” region , is presented in fig2 . the second type of sequence representing the majority of the clones sequences is represented by the sequence msrv - 2b presented in fig5 and identified by seq id no11 . the region amplified between the pcr primers is homologous , apart from a single base , with the msrv2 - a sequence ( seq id no10 according to fig1 ) lying inside the pcr primers , described in example 1 . the differences observed in the sequences corresponding to the pcr primers are explained by the use of degenerate primers in mixture form used under different technical conditions . the sequences msrv - 2a ( seq id no10 ) and msrv - 2b ( seq id no11 ) are manifestly homologous , or even identical , derived from the same organism and sufficiently divergent from the retroviral sequences already described in the data banks for it to be suggested that the sequence region in question belongs to a new infective agent , designated msrv - 2 . this infective agent would be , in principle , on the basis of the analysis of the first sequences obtained , related to a retrovirus but , in view of the technique used for obtaining this sequence , it could also be a dna virus whose genome codes for an enzyme which incidentally possesses reverse transcriptase activity , as is the case , for example , with the hepatitis b virus , hbv ( 33 ). furthermore , the random nature of the degenerate primers used for this pcr amplification technique may very well have permitted , as a result of unforeseen sequence homologies or of conserved sites in the gene for a related enzyme , the amplification of a nucleic acid originating from a prokaryotic or eukaryotic pathogenic and / or coinfective agent ( protist ). obtaining clones designated msrv - 1b and msrv - 2b , defining a family msrv - 1 and msrv2 , by “ nested ” pcr amplification of the conserved pol regions of retroviruses on preparations of b lymphocytes from a new case of ms the same pcr technique , modified according to the technique of shih ( 33 ), was used to amplify and sequence the rna nucleic acid material present in a purified fraction of virions at the peak of “ lm7 - like ” reverse transcriptase activity on a sucrose gradient according to the technique described by h . perron ( 34 ), and according to the protocols mentioned in example 2 , from a spontaneous lymphoblastoid line obtained by self - immortalization in culture of b lymphocytes from an ms patient who was seropositive for the epstein - barr virus ( ebv ), after setting up the blood lymphoid cells in culture in a suitable culture medium containing a suitable concentration of cyclosporin a . a representation of the reverse transcriptase activity in the sucrose fractions taken from a purification gradient of the virions produced by this line is presented in fig6 . similarly , the culture supernatants of a b line obtained under the same conditions from a control free from multiple sclerosis were treated under the same conditions , and the assay of reverse transcriptase activity in the sucrose gradient fractions proved negative throughout ( background ), and is presented in fig7 . fraction 3 of the gradient corresponding to the ms b line and the same fraction without reverse transcriptase activity of the non - ms control gradient were analyzed by the same rt - pcr technique as before , derived from shih ( 33 ), followed by the same steps of cloning and sequencing as described in examples 1 and 2 . it is particularly noteworthy that the msrv - 1 and msrv - 2 type sequences are to be found only in the material associated with a peak of “ lm7 - like ” reverse transcriptase activity originating from the ms b lymphoblastoid line . these sequences were not to be found with the material from the control ( non - ms ) b lymphoblastoid line in 26 recombinant clones taken at random . only mo - mulv type contaminant sequences , originating from the commercial reverse transcriptase used for the cdna synthesis step , and sequences without any particular retroviral analogy were to be found in this control , as a result of the “ consensus ” amplification of homologous polymerase sequences which is produced by this pcr technique . furthermore , the absence of a concentrated target which competes for the amplification reaction in the control sample permits the amplification of dilute contaminants . the difference in results is manifestly highly significant ( chi - squared , p & lt ; 0 . 001 ). obtaining a clone psj17 , defining a retrovirus msrv - 1 , by reaction of endogenous reverse transcriptase with a virion preparation originating from the pli - 2 line this approach is directed towards obtaining reverse - transcribed dna sequences from the supposedly retroviral rna in the isolate using the reverse transcriptase activity present in this same isolate . this reverse transcriptase activity can theoretically function only in the presence of a retroviral rna linked to a primer trna or hybridized with short strands of dna already reverse - transcribed in the retroviral particles ( 39 ). thus , the obtaining of specific retroviral sequences in a material contaminated with cellular nucleic acids was optimized according to these authors by means of the specific enzymatic amplification of the portions of viral rnas with a viral reverse transcriptase activity . to this end , the authors determined the particular physicochemical conditions under which this enzymatic activity of reverse transcription on rnas contained in virions could be effective in vitro . these conditions correspond to the technical description of the protocols presented below ( endogenous rt reaction , purification , cloning and sequencing ). the molecular approach consisted in using a preparation of concentrated but unpurified virion obtained from the culture supernatants of the pli - 2 line , prepared according to the following method : the culture supernatants are collected twice weekly , precentrifuged at 10 , 000 rpm for 30 minutes to remove cell debris and then frozen at − 80 ° c . or used as they are for the following steps . the fresh or thawed supernatants are centrifuged on a cushion of 30 % glycerol - pbs at 100 , 000 g ( or 30 , 000 rpm in a type 45 t lkb - hitachi rotor ) for 2 h at 4 ° c . after removal of the supernatant , the sedimented pellet is taken up in a small volume of pbs and constitutes the fraction of concentrated but unpurified virion . this concentrated but unpurified viral sample was used to perform a so - called endogenous reverse transcription reaction as will now be described : a volume of 200 μl of virion purified according to the protocol described above , and containing a reverse transcriptase activity of approximately 1 - 5 million dpm , is thawed at 37 ° c . until a liquid phase appears , and then placed on ice . a 5 - fold concentrated buffer was prepared with the following components : 500 mm tris - hcl ph 8 . 2 ; 75 mm nacl ; 25 mm mgcl 2 ; 75 mm dtt and 0 . 10 % np 40 . 100 μl of 5 × buffer + 25 μl of a 100 mm solution of datp + 25 μl of a 100 mm solution of dttp + 25 μl of a 100 mm solution of dgtp + 25 μl of a 100 mm solution of dctp + 100 μl of sterile distilled water + 200 μl of the virion suspension ( rt activity of 5 million dpm ) in pbs were mixed and incubated at 42 ° c . for 3 hours . after this incubation , the reaction mixture is added directly to a buffered phenol / chloroform / isoamyl alcohol mixture ( sigma ref . p 3803 ); the aqueous phase is collected and one volume of sterile distilled water is added to the organic phase to re - extract the residual nucleic acid material . the collected aqueous phases are combined , and the nucleic acids contained are precipitated by adding 3m sodium acetate ph 5 . 2 to { fraction ( 1 / 10 )} volume + 2 volumes of ethanol + 1 μl of glycogen ( boehringer - mannheim ref . 910 393 ) and placing the sample at − 20 ° c . for 4 h or overnight at + 4 ° c . the precipitate obtained after centrifugation is then washed with 70 % ethanol and resuspended in 60 ml of distilled water . the products of this reaction were then purified , cloned and sequenced according to the protocol which will now be described : blunt - ended dnas with unpaired adenines at the ends were generated : a “ filling - in ” reaction was first performed : 25 μl of the previously purified dna solution were mixed with 2 μl of a 2 . 5 mm solution containing , in equimolar amounts , datp + dgtp + dttp + dctp / 1 μl of t4 dna polymerase ( boehringer - mannheim ref . 1004 786 )/ 5 μl of 10 ×“ incubation buffer for restriction enzyme ” ( boehringer - mannheim ref . 1417 975 )/ 1 μl of a 1 % bovine serum albumin solution / 16 μl of sterile distilled water . this mixture was incubated for 20 minutes at 11 ° c . 50 μl of te buffer and 1 μl of glycogen ( boehringer - mannheim ref . 901 393 ) were added thereto before extraction of the nucleic acids with phenol / chloroform / isoamyl alcohol ( sigma ref . p 3803 ) and precipitation with sodium acetate as described above . the dna precipitated after centrifugation is resuspended in 10 μl of 10 mm tris buffer ph 7 . 5 . 5 μl of this suspension were then mixed with 20 μl of 5 × taq buffer , 20 μl of 5 mm datp , 1 μl ( 5 u ) of taq dna polymerase ( amplitaq ™) and 54 μl of sterile distilled water . this mixture is incubated for 2 h at 75 ° c . with a film of oil on the surface of the solution . the dna suspended in the aqueous solution drawn off under the film of oil after incubation is precipitated as described above and resuspended in 2 μl of sterile distilled water . the dna obtained was inserted into a plasmid using the ta cloning kit ™. the 2 μl of dna solution were mixed with 5 μl of sterile distilled water , 1 μl of a 10 - fold concentrated ligation buffer “ 10 × ligation buffer ”, 2 μl of “ pcr ™ vector ” ( 25 ng / ml ) and 1 μl of “ ta dna ligase ”. this mixture was incubated overnight at 12 ° c . the following steps were carried out according to the instructions of the ta cloning ® kit ( british biotechnology ). at the end of the procedure , the white colonies of recombinant ( white ) bacteria were picked out in order to be cultured and to permit extraction of the plasmids incorporated according to the so - called “ miniprep ” procedure ( 36 ). the plasmid preparation from each recombinant colony was cut with a suitable restriction enzyme and analyzed on agarose gel . plasmids possessing an insert detected under uv light after staining the gel with ethidium bromide were selected for sequencing of the insert , after hybridization with a primer complementary to the sp6 promoter present on the cloning plasmid of the ta cloning kit ®. the reaction prior to sequencing was then performed according to the method recommended for the use of the sequencing kit “ prism ready reaction kit dye deoxyterminator cycle sequencing kit ” ( applied biosystems , ref . 401384 ), and automatic sequencing was carried out with an applied biosystems “ model 373 a automatic sequencer ” apparatus according to the manufacturer &# 39 ; s instructions . discriminating analysis on the computerized data banks of the sequences cloned form the dna fragments present in the reaction mixture enabled a retroviral type sequence to be revealed . the corresponding clone psj17 was completely sequenced , and the sequence obtained , presented in fig8 and identified by seq id no9 , was analyzed using the “ geneworks ®” software on the updated “ genebank ®” data banks . an identical sequence already described could not be found by analysis of the data banks . only a partial homology with some known retroviral elements was to be found . the most useful relative homology relates to an endogenous retrovirus designated erv - 9 , or hserv - 9 , depending on the references ( 40 ). pcr amplification of the nucleic acid sequence contained between the 5 ′ region defined by the clone “ pol msrv - 1b ” and the 3 ′ region defined by the clone psj17 five oligonucleotides , m001 , m002 - a , m003 - bcd , p004 and p005 , were defined in order to amplify the rna originating from purified pol - 2 virions . control reactions were performed so as to check for the presence of contaminants ( reaction with water ). the amplification consists of an rt - pcr step according to the protocol described in example 2 , followed by a “ nested ” pcr according to the pcr protocol described in the document ep - a - 0569272 . in the first rt - pcr cycle , the primers m001 and p004 or p005 are used . in the second pcr cycle , the primers m002 - a or m003 - bcd and the primer p004 are used . the primers are positioned as follows : the “ nested ” amplification product obtained , and designated m003 - p004 , is presented in fig9 and corresponds to the sequence seq id no8 . amplification and cloning of a portion of the msrv - 1 retroviral genome using a sequence already identified , in a sample of virus purified at the peak of reverse transcriptase activity a pcr technique derived from the technique published by frohman ( 41 ) was used . the technique derived makes it possible , using a specific primer at the 3 ′ end of the genome to be amplified , to elongate the sequence towards the 5 ′ region of the genome to be analyzed . this technical variant is described in the documentation of the firm “ clontech laboratories inc ., ( palo - alto , calif ., usa ) supplied with its product “ 5 ′- amplifinder ™ race kit ”, which was used on a virion fraction purified as described above . the specific 3 ′ primers used in the kit protocol for the synthesis of the cdna and the pcr amplification are , respectively , complementary to the following msrv - 1 sequences : the products originating from the pcr were purified after purification on agarose gel according to conventional methods ( 36 ), and then resuspended in 10 ml of distilled water . since one of the properties of taq polymerase consists in adding an adenine at the 3 ′ end of each of the two dna strands , the dna obtained was inserted directly into a plasmid using the ta cloning kit ™ ( british biotechnology ). the 2 μl of dna solution were mixed with 5 μl of sterile distilled water , 1 μl of a 10 - fold concentrated ligation buffer “ 10 × ligation buffer ”, 2 μl of “ pcr ™ vector ” ( 25 ng / ml ) and 1 μl of “ ta dna ligase ”. this mixture was incubated overnight at 12 ° c . the following steps were carried out according to the instructions of the ta cloning ® kit ( british biotechnology ). at the end of the procedure , the white colonies of recombinant ( white ) bacteria were picked out in order to be cultured and to permit extraction of the plasmids incorporated according to the so - called “ mini - prep ” procedure ( 36 ). the plasmid preparation from each recombinant colony was cut with a suitable restriction enzyme and analyzed on agarose gel . plasmids possessing an insert detected under uv light after staining the gel with ethidium bromide were selected for sequencing of the insert , after hybridization with a primer complementary to the sp6 promoter present on the cloning plasmid of the ta cloning kit ®. the reaction prior to sequencing was then performed according to the method recommended for the use of the sequencing kit “ prism ready reaction kit dye deoxyterminator cycle sequencing kit ” ( applied biosystems , ref . 401384 ), and automatic sequencing was carried out with an applied biosystems “ model 373 a automatic sequencer ” apparatus according to the manufacturer &# 39 ; s instructions . this technique was applied first to two fractions of virion purified as described below on sucrose from the “ pol - 2 ” isolate produced by the pli - 2 line on the one hand , and from the ms7pg isolate produced by the lm7pc line on the other hand : the culture supernatants are collected twice weekly , precentrifuged at 10 , 000 rpm for 30 minutes to remove cell debris and then frozen at − 80 ° c . or used as they are for the following steps . the fresh or thawed supernatants are centrifuged on a cushion of 30 % glycerol - pbs at 100 , 000 g ( or 30 , 000 rpm in a type 45 t lkb - hitachi rotor ) for 2 h at 4 ° c . after removal of the supernatant , the sedimented pellet is taken up in a small volume of pbs and constitutes the fraction of concentrated but unpurified virions . the concentrated virus is then applied to a sucrose gradient in sterile pbs buffer ( 15 to 50 % weight / weight ) and ultracentrifuged at 35 , 000 rpm ( 100 , 000 g ) for 12 h at + 4 ° c . in a swing - out rotor . 10 fractions are collected , and 20 μl are withdrawn from each fraction after homogenization to assay the reverse transcriptase activity therein according to the technique described by h . perron ( 24 ). the fractions containing the peak of “ lm7 - like ” rt activity are then diluted in sterile pbs buffer and ultracentrifuged for one hour at 35 , 000 rpm ( 100 , 000 g ) to sediment the viral particles . the pellet of purified virion thereby obtained is then taken up in a small volume of a buffer which is appropriate for the extraction of rna . the cdna synthesis reaction mentioned above is carried out on this rna extracted from purified extracellular virion . pcr amplification according to the technique mentioned above enabled the clone f1 - 11 to be obtained , whose sequence , identified by seq id no2 , is presented in fig1 . this clone makes it possible to define , with the different clones previously sequenced , a region representative of the “ pol ” gene of the msrv - 1 retrovirus , as presented in fig1 . this sequence , designated seq id no1 , is reconstituted from different clones overlapping one another at their ends , correcting the artifacts associated with the primers and with the amplification or cloning techniques which would artificially interrupt the reading frame of the whole . in fig1 , the potential reading frame with its translation into amino acids is presented below the nucleic acid sequence . capture , amplification and cloning of a portion of the msrv - 2 genome using a sequence already identified , in a culture infected with msrv - 2 the supernatants of a cell culture expressing “ lm7 - like ” reverse transcriptase activity similar to that described by h . perron ( 24 ) were collected regularly over several weeks and stored frozen at − 80 ° c . after adding 10 % of glycerol . the set of supernatants was then thawed so as to concentrate the infective particles by ultracentrifugation and to purify them by centrifugation to equilibrium on a sucrose gradient ; the reverse transcriptase activity was then measured in the different fractions collected on the gradient according to the methodology described by h . perron ( 34 ). the different fractions representing the peak of reverse transcriptase activity were pooled so as to extract the nucleic acids therefrom according to a protocol intended for the purification of rna ( 35 ), but the nucleic acids extracted were not treated with dnase . a pcr amplification derived from the technique described by shih ( 33 ) was performed directly on this nucleic acid sample not treated with dnase , according to an rna amplification method as described in the document ep - a - 0 , 569 , 272 , in a total volume of 100 μl containing 200 ng of rna , 1 μl of rna guard and 33 μmol of each mixture of primers ( mop ) which are described by shih ( 33 ) and identical to those used for the direct ( dna ) pcr ; 0 . 25 mm each dntp , 10 μl of 10 × buffer , 2 . 5 u of taq enzyme and 0 . 4 μl of rt enzyme ( rt - amv ; 10 u ) are also added to the samples . the amplification cycles are carried out as follows : denaturation of the rna 65 ° c ./ 10 minutes , synthesis of the cdna 50 ° c ./ 8 minutes , then the cycles are identical to those of the pcr described by shih ( 33 ). control reactions were performed so as to check for the absence of contaminants ( reaction with water ). the products were analyzed on 10 % acrylamide gel . the samples amplified by rt - pcr were then cloned and sequenced according to the techniques described in example 1 . the majority of the clones sequenced from the rt - pcr product corresponds to the msrv - 2a sequence and its equivalent msrv - 2b described above in examples 1 to 3 . moreover , after removal of the artifactual sequences , the other clones sequenced prove to correspond to msrv - 1 type sequences as are described in examples 1 to 3 . after verification of the sequences present in this nucleic acid material originating from these purified fractions containing infective particles , at least a part of which is associated with reverse transcriptase activity , the remaining nucleic acid material was used to perform a specific capture of nucleic acids carrying the msrv2 sequence previously identified and described in examples 1 to 3 . in a prior step , the genetic material carrying the msrv2 sequence was amplified by a one - directional pcr technique of 50 cycles using a single primer . this primer is coupled to a biotin molecule at its 3 ′ end , permits one - directional amplification from 3 ′ to 5 ′ and corresponds to the following sequence identified under seq id no38 : thereafter , capture was performed in solution with magnetic beads coupled to avidin ( dynabeads ®) according to the instructions of the manufacturer ( dynal ) and , after a series of washes at room temperature enabling nucleic acids not coupled to a biotin to be removed , a pcr was performed directly on these washed beads with a specific primer at the 3 ′ end and a primer at the 5 ′ end provided by a solution of oligonucleotide of 10 bases ( 10 - mer ) with a random sequence . the specific amplification primer oriented from 3 ′ to 5 ′ corresponds to the sequence identified by seq id no13 : the pcr performed at 35 ° c . over 40 cycles with these primers enabled the genetic material specifically biotinylated by the first pcr step and captured on the dynabeads ® beads to be amplified . after cloning with the “ ta cloning ” kit of the dna amplified by this second pcr step and sequencing of the recombinant clones , according to the techniques described in example 1 , a sequence of 748 base pairs was obtained . this nucleic acid sequence seq id no12 is presented in fig1 . this elongated sequence will be designated hereafter msrv - 2el1 . the reverse sequence complementary to the primer seq id no13 is present at the 3 ′ end and is boxed in fig1 . upstream of this primer , the sequence already identified in the msrv - 2a and msrv - 2b clones is to be found . the translation of this sequence into amino acids according to the 6 possible reading frames is presented in fig1 . an alignment of the msrv2 - a sequence ( seq id no10 ) with the msrv - 2el1 sequence ( seq id no12 ) is presented in fig1 . it will be noted that the msrv - 2a sequence is strictly identical to the elongated sequence , apart from a few differences in the region corresponding to the degenerate primers used for obtaining msrv - 2a . this region is underlined in this figure ; moreover , the hybridization region of the primer seq id no13 ( apart from the cloning tail ) is boxed , that of the primer seq id no14 is presented between square brackets . the true sequence of the msrv - 2 genome in this region is probably that of msrv - 2el1 , where it has not been imposed by hybridized primers having low stringency as is the case for msrv - 2a ( and msrv - 2b likewise ). the msrv - 2el1 sequence hence corresponds to a new sequenced region of the msrv - 2 genome . this was verified using new pcr primers defined in msrv - 2el1 and msrv - 2a , which permitted a specific amplification on the nucleic acids used for the cloning described in this example . the examples which follow present different results of specific msrv2 amplifications which confirm the relationship with the presence of corresponding infective agent in the cell cultures described , to permit the isolation of an lm7 type virus ( 24 ), and also , in vivo , in patients suffering from ms . the result of interrogation of the genebank ® data bank , updated in august 1994 , with the msrv - 2el1 sequence does not show any significant homology with genetic sequences known to date . however , the interrogation of the possible translations into amino acids according to the 6 potential reading frames of this msrv - 2el1 sequence shows partial homologies with bacterial , viral or cellular sequences . the absence of pcr amplification with specific primers on normal human dna shows that the sequence in question is not one of cellular origin . msrv - 2 is hence an infective agent exogenous to man . however , the degenerate nature of the mixtures of primers used according to variants of the technique described by shih ( 33 ), which enabled the first sequence elements designated msrv - 2a and msrv - 2b to be identified , may have permitted the unforeseen amplification of a genome not belonging to a retrovirus , or even to a gene coding for an rna - dependent dna polymerase . the almost invariable co - detection of msrv - 1 in cultures originating from ms and expressing reverse transcriptase activity may be explained by a pathological association between two different agents , at least one of which is a retrovirus ( msrv - 1 ). the detection in patients of these two types of sequence described in the examples which follow corroborates a pathological association . however , only one of these elements may suffice to explain the pathology induced in ms . detection of specific msrv - 2 sequences in different samples of human cells originating from patients suffering from ms or from controls the msrv - 2el1 sequence ( seq id no12 ) enabled several pairs of oligonucleotide primers which could be used for the amplification of specific dna or rna by the pcr technique to be defined . the primers defined below enabled a specific detection of the msrv - 2 genome in different human cells to be carried out by an rt - pcr step according to an rna amplification method as described in the document ep - a - 0 , 569 , 272 . the pcr is performed according to a succession of 35 cycles linking together , after the cdna synthesis step , 1 min at 94 ° c ., 1 min at 54 ° c . and 1 min at 72 ° c . the total rna extracted from different cell types ( 35 ), without dnase treatment , was used in this rt - pcr reaction . fig1 presents the results of pcr using a photograph under ultraviolet light of an ethidium bromide - impregnated agarose gel , in which an electrophoresis of pcr amplification products applied separately to the different wells was performed . well number 1 contains a mixture of dna molecular weight markers , and wells 2 to 9 represent , in order , the products amplified from the total rnas of the following cells : 7 — cells originating from a mixture of b lymphoblastoid lines derived from the peripheral blood of different patients suffering from ms ; 8 — cells originating from a b lymphoblastoid line derived from the peripheral blood of a patient suffering from ms ; the existence of a band of specific dna of approximately 700 base pairs , corresponding to the expected size , which is amplified in the samples originating from patients suffering from ms ( lm7pc , pli2 , b lymphocyte lines ) and not in the cells tested originating from controls not suffering from ms ( mrc5 , blood mononuclear cells and medulloblastoma cells ), can be seen . detection of specific msrv - 1 and msrv - 2 sequences in different samples of plasma originating from patients suffering from ms or from controls a pcr technique similar to the one described in example 8 was used to detect the msrv - 1 and msrv - 2 genomes in plasmas obtained after taking blood samples from patients suffering from ms and from non - ms controls onto edta . extraction of the rnas from plasma was performed according to a technique described by p . chomzynski ( 35 ), after adding one volume of buffer containing guanidinium thiocyanate to 1 ml of plasma stored frozen at − 80 ° c . after collection . for msrv - 2 , the pcr was performed under the same conditions and with the same primers as those described in example 8 . however , similar results were also obtained with the following pcr primers in two successive amplifications by “ nested ” pcr on samples of nucleic acids not treated with dnase . the primers used for this first step of 40 cycles with a hybridization temperature of 48 ° c . are the following : 5 ′ gccgatatcacccgccatgg 3 ′, corresponding to a 5 ′ msrv - 2 pcr primer , for a first pcr on patients &# 39 ; sample , 5 ′ gcatccggcaactgcacg 3 ′, corresponding to a 3 ′ msrv - 2 pcr primer , for a first pcr on patients &# 39 ; sample after this step , 10 μl of the amplification product are taken and used to carry out a second , so - called “ nested ” pcr amplification with primers located within the region already amplified . this second step takes place over 35 cycles , with a primer hybridization (“ annealing ”) temperature of 50 ° c . the reaction volume is 100 μl . 5 ′ cgcgatgctggttggagagc 3 ′, corresponding to a 5 ′ msrv - 2 pcr primer , for a nested pcr on patients &# 39 ; sample , 5 ′ tctccactccgaatattccg 3 ′, corresponding to a 3 ′ msrv - 2 pcr primer , for a nested pcr on patients &# 39 ; sample . for msrv - 1 , the amplification was performed in two steps . furthermore , the nucleic acid sample is treated beforehand with dnase , and a control pcr without rt ( amv reverse transcriptase ) is performed on the two amplification steps so as to verify that the rt - pcr amplification comes exclusively from the msrv - 1 rna . in the event of a positive control without rt , the initial aliquot sample of rna is again treated with dnase and amplified again . the protocol for treatment with dnase lacking rnase activity is as follows : the extracted rna is aliquoted in the presence of “ rnase inhibitor ” ( boehringer - mannheim ) in water treated with depc at a final concentration of 1 μg in 10 μl ; to these 10 μl , 1 μl of “ rnase - free dnase ” ( boehringer - mannheim ) and 1 . 2 μl of ph 5 buffer containing 0 . 1 m sodium acetate and 5 mm mgso 4 are added ; the mixture is incubated for 15 min at 20 ° c . and brought to 95 ° c . for 1 . 5 min in a “ thermocycler ”. the first msrv - 1 rt - pcr step is performed according to a variant of the rna amplification method as described in patent application no . ep 0 , 569 , 272 a1 . in particular , the cdna synthesis step is performed at 42 ° c . for one hour ; the pcr amplification takes place over 40 cycles , with a primer hybridization (“ annealing ”) temperature of 53 ° c . the reaction volume is 100 μl . after this step , 10 μl of the amplification product are taken and used to carry out a second , so - called “ nested ” pcr amplification with primers located within the region already amplified . this second step takes place over 35 cycles , with a primer hybridization (“ annealing ”) temperature of 53 ° c . the reaction volume is 100 μl . fig1 presents the results of pcr in the form of a photograph under ultraviolet light of an ethidium bromide - impregnated agarose gel , in which an electrophoresis of the pcr amplification products applied separately to the different wells was performed . well number 8 contains a mixture of dna molecular weight markers , and wells 1 to 7 represent , in order , the products amplified from the total rnas of plasmas originating from 4 healthy controls free from ms ( wells 1 to 4 ) and from 3 patients suffering from ms at different stages of the disease ( wells 5 to 7 ). in this series , msrv - 2 nucleic acid material is detected in the plasma of one case of ms out of the 3 tested , and in none of the 4 control plasmas . other results obtained on more extensive series confirm these results . fig1 shows the result of specific amplification by msrv - 1 “ nested ” rt - pcr : well no . 1 contains the pcr product produced with water alone , without the addition of amv reverse transcriptase ; well no . 2 contains the pcr product produced with water alone , with addition of amv reverse transcriptase ; well number 3 contains a mixture of dna molecular weight markers ; wells 4 to 13 contain , in order , the products amplified from the total rnas extracted from sucrose gradient fractions ( collected in a downward direction ), on which gradient a pellet of virion originating from a supernatant of a culture infected with msrv - 1 and msrv - 2 was centrifuged to equilibrium according to the protocol described by perron ( 34 ); to well 14 nothing was applied ; to wells 15 to 17 , the amplified products of rna extracted from plasmas originating from 3 different patients suffering from ms at different stages of the disease were applied . the msrv - 1 retroviral genome is indeed to be found in the sucrose gradient fraction containing the peak of reverse transcriptase activity measured according to the technique described by h . perron ( 24 ), with a very strong intensity ( fraction 5 of the gradient , deposited in well no . 8 ). a slight amplification has taken place in the first fraction ( well no . 4 ), probably corresponding to rna released by lysed particles which floated at the surface of the gradient ; similarly , aggregated debris sedimented in the last fraction ( tube bottom ), carrying with it a few copies of the msrv - 1 genome which have given rise to an amplification of low intensity . of the 3 ms plasmas tested in this series , msrv - 1 rna turned up in one case , producing a very intense amplification ( well no . 17 ). in this series , the msrv - 1 retroviral rna genome , probably corresponding to particles of extracellular virus present in the plasma in extremely small numbers , was detected by “ nested ” rt - pcr in one case of ms out of the 3 tested . other results obtained on more extensive series confirm these results . furthermore , the specificity of the sequences amplified by these pcr techniques may be verified and evaluated by the “ elosa ” technique as described by f . mallet ( 42 ) and in the document fr - 2 , 663 , 040 . for msrv - 1 , the products of the nested pcr described above may be tested in two elosa systems enabling a consensus a and a consensus b + c + d of msrv - 1 to be detected separately , corresponding to the subfamilies described in example 2 and fig2 and 4 . in effect , the sequences closely resembling the consensus b + c + d are to be found essentially in the rna samples originating from msrv - 1 virions purified from cultures or amplified in extracellular biological fluids of ms patients , whereas the sequences closely resembling the consensus a are essentially to be found in normal human cellular dna . the elosa / msrv - 1 system for the capture and specific hybridization of the pcr products of the subfamily a uses a capture oligonucleotide cpv1a with an amine bond at the 5 ′ end and a biotinylated detection oligonucleotide dpv1a having as their sequence , respectively : 5 ′ gatctaggccacttctcaggtccags 3 ′, corresponding to the elosa capture oligonucleotide for the products of msrv - 1 nested pcr performed with the primers identified by seq id no16 and seq id no17 , optionally followed by amplification with the primers identified by seq id no18 and seq id no19 on samples from patients . 5 ′ catctitttggicaggcaitagc 3 ′ corresponding to the elosa detection oligonucleotide for the subfamily a of the products of msrv - 1 nested pcr performed with the primers identified by seq id no16 and seq id no17 , optionally followed by amplification with the primers identified by seq id no18 and seq id no19 on samples from patients . the elosa / msrv - 1 system for the capture and specific hybridization of the pcr products of the subfamily b + c + d uses the same biotinylated detection oligonucleotide dpv1a and a capture oligonucleotide cpv1b with an amine bond at the 5 ′ end having as its sequence : 5 ′ cttgagccagttctcatacctgga 3 ′, corresponding to the elosa capture oligonucleotide for the subfamily b + c + d of the products of msrv - 1 nested pcr performed with the primers identified by seq id no16 and seq id no17 , optionally followed by amplification with the primers identified by seq id no18 and seq id no19 on samples from patients . this elosa detection system enabled it to be verified that none of the pcr products thus amplified from dnase - treated plasmas of ms patients contained a sequence of the subfamily a , and that all were positive with the consensus of the subfamilies b , c and d . for msrv - 2 , a similar elosa technique was evaluated on isolates originating from infected cell cultures , using the following pcr amplification primers , 5 ′ agtgytrccmcarggcgctgaa 3 ′, corresponding to a 5 ′ msrv - 2 pcr primer , for pcr on sample from cultures , 5 ′ gmggccagcagsakgtcatcca 3 ′, corresponding to a 3 ′ msrv - 2 pcr primer , for pcr on sample from cultures , and the capture oligonucleotides with an amine bond at the 5 ′ end cpv2 and the biotinylated detection oligonucleotide dpv2 having as their respective sequences : 5 ′ ggatgccgcctatagcctctac 3 ′, corresponding to an elosa capture oligonucleotide for the products of msrv - 2 pcr performed with the primers seq id no34 and seq id no35 , or optionally with the degenerate primers defined by shih ( 33 ), 5 ′ aagcctatcgcgtgcagttgcc 3 ′, corresponding to an elosa detection oligonucleotide for the products of msrv - 2 pcr performed with the primers seq id no34 and seq id no35 , or optionally with the degenerate primers defined by shih ( 33 ) this pcr amplification system with a pair of primers different from those which were described previously for amplification on the samples from patients made it possible to confirm the infection with msrv - 2 of in vitro cultures and of samples of nucleic acids used for the molecular biology studies . all things considered , our first results of pcr detection of the genome of pathogenic and / or infective agents , it is possible that free “ virus ” may circulate in the blood stream of patients in an acute , virulent phase , outside the nervous system . this is compatible with the almost invariable presence of “ gaps ” in the blood - brain barrier of patients in an active phase of ms . it is thus already conceivable , as a result of the discoveries made and the methods developed by the inventors , to carry out a diagnosis of msrv - 1 and / or msrv - 2 infection and / or reactivation and to evaluate a therapy in ms on the basis of its efficacy to “ negative ” the detection of these agents in the patients &# 39 ; biological fluids . furthermore , early detection in individuals not yet displaying neurological signs of ms could make it possible to institute a treatment which would be all the more effective with respect to the subsequent clinical course for the fact that it would precede the lesion stage which corresponds to the onset of neurological disorders . now , at the present time , a diagnosis of ms cannot be established before a symptomatology of neurological lesions has set in , and hence no treatment is instituted before the emergence of a clinical picture suggestive of lesions of the central nervous system which are already significant . the diagnosis of an msrv - 1 and / or msrv - 2 infection and / or reactivation in man is hence of decisive importance , and the present invention provided the means of doing this . it is thus possible , apart from carrying out a diagnosis of msrv - 1 and / or msrv - 2 infection and / or reactivation , to evaluate a therapy in ms on the basis of its efficacy to “ negative ” the detection of these agents in the patients &# 39 ; biological fluids . 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