NZ303694A - Use of viral material comprising msrv-1 or msrv-2 to prepare diagnostic, prophylactic and therapeutic compositions - Google Patents

Description

New Zealand No. 303694 International No. PCT/FR96/00360 TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates: 09.03.1995; Complete Specification Filed: 07.03.1996 Classification^) C12N15/48.11; C12N7/02; C07K14/15; C12Q1/68; G01N33/569; A61K39/21; A61K35/76 Publication date: 24 November 1997 Journal No.: 1422 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of invention: Rheumatoid arthritis-related virus MSRV-1 and pathogenic and/or infective agent MSRV-2 Name, address and nationality of applicant(s) as in international application form: BIO MERIEUX, 69280, Marcy L-etoile, France New Zealand No. International No. 303694 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Rheumatoid arthritis-related virus MSRV-1 and pathogenic and/or infective agent MSRV-2 Name, address and nationality of applicant(s) as in international application form: BIO MERIEUX, of 69280, Marcy L-etoile, France 3 6 9 4 WO 96/28552 - 1 - PCT/FR96/00360 MSRV-1 VIRUS AND PATHOGENIC AMD/OR INFECTING BISRV-2 AGENT ASSOCIATED WITH RHEUMATOID ARTHRITIS Rheumatoid arthritis (RA) is the most common of the human inflammatory rheumatisms and is of particularly 5 high prevalence: 3% (1). Diagnosis is difficult at the start: its clinical heterogeneity and the extra-articular symptoms associated with sometimes considerable immunological anomalies complicate the picture. This is a disease whose aetiology is still unknown, which is 10 classed in the category of "autoimmune" diseases on account of the existence of exacerbated immunological reactions against auto-antigens.

A viral and/or bacterial aetiology of RA has often been put forward, but the search for a causal agent 15 of RA has hitherto been unsuccessful (2).

The concepts defining RA as an autoimmune disease and those which support a viral, or even bacterial, aetiological hypothesis may now be combined in the understanding of the various mechanisms by which a micro-2 0 organism can induce an autoimmune reaction in the infected host, as has been described by Fujinami R.S. and Oldstone M.B.A. (3). Molecules of bacterial or viral origin, or even of endogenous retroviral origin, are known to possess so-called superantigenic properties (4, 25 5). Their particular properties of direct stimulation of T lymphocytes, which are specific for different antigens, by binding with the V region of certain "T" receptors, have evoked the hypothesis that such molecules are intimately associated with the aetiopathogenic process of 30 autoimmune pathologies (6). However, no specific bacterial or viral pathogenic agent, and possibly superantigen producer, has yet been clearly associated with rheumatoid arthritis.

The Applicant's work in the search for an aeti-35 ology of RA has now led to the discovery of the existence of two pathological and/or infective agents associated, independently or together, with the pathological states of RA.

The techniques for culturing and detecting 2 - 3 6 94 retroviral material which are used in the work performed by the Applicant on an agent associated with multiple sclerosis (MS) and described in French patent applications 92 04322, 92 13447, 92 13443, 92 01529, 94 01530, 5 94 01531 and 94 01532 and in the publication by H. Perron et al. (7) (the content of which is included in the present description by way of reference) have made it possible to demonstrate, entirely unexpectedly, the similarity between the agents associated with MS and 10 those which form the subject of the present invention, associated with RA.

Given that, in RA, the process takes place in the joints and more particularly involves the synovial membrane, cultures were made of cells derived from 15 synovial fluid punctured from the affected joints of patients suffering from RA. Using such a culture, it was possible to obtain cells of fibroblast type which proliferated in vitro, thereby allowing a few runs to be carried out. The corresponding culture media were used 20 for a study of reverse transcriptase activity associated with infective particles concentrated by ul tr a can tr if ligation and then purified on an isopycnic gradient. The use of the detecting conditions developed for the LM7 strain derived from MS (7) has made it possible to detect 25 significant reverse transcriptase activity in a few distinct fractions of such a gradient. Thereafter, the analysis of the nucleic acid sequences amplified in these fractions by the PCR (polymerase chain reaction) technique, with degenerate primers similar to the consensus 30 sequences of the enzymes with reverse transcriptase activity (8), revealed the presence of significant sequences identical to the sequences of the pathogenic and/or infectious agents MSRV1 and MSRV2, previously identified in cultures derived from cases of multiple 35 sclerosis.

Thus, the various subjects of the present invention are as follows: i) the use of a viral material, in the purified or isolated state, possessing reverse transcriptase 3 0 6 8 y activity, related to a family of endogenous retroviral elements, as derived from a viral strain possessing reverse transcriptase activity, chosen from the strains referred to respectively as POL-2, filed on 22.07.1992 5 with the ECACC under the access number V92072202, and MS7PG filed on 08.01.93 with the ECACC under the access number V93010816, and from the variant strains thereof, the variant strains consisting of viruses conqprising at least one antigen which is recognized by at least one 10 antibody directed against at least one antigen corresponding to either of the viruses of the abovementioned viral strains POL-2 and MS7PG, in order to obtain a diagnostic, prophylactic or therapeutic composition for detecting, preventing or treating an infection by the 15 said viral material or a reactivation of the said viral material, associated with rheumatoid arthritis, ii) the use of a viral material, in the purified or isolated state, possessing reverse transcriptase activity, related to a family of endogenous retroviral elements, as produced by a cell line chosen from the lines referred to respectively as PLI-2 filed on 22.07.1992 with the ECACC under the access number 92072201, and LM7PC filed on 08.01.93 with the ECACC under the access number 93010817, or by any infected cell 25 culture capable of producing a virus comprising at least one antigen which is recognized by at least one antibody directed against at least one antigen corresponding to either of the viruses produced by the abovementioned lines PLI-2 and LM7PC, in order to obtain a diagnostic, 30 prophylactic or therapeutic composition for detecting, preventing or treating an infection by the said viral material or a reactivation of the said viral material, associated with rheumatoid arthritis, iii) the use of a viral material whose genome 35 comprises a nucleotide sequence chosen from SEQ ID NOl, SEQ ID N02, SEQ ID N03, SEQ ID N04, SEQ ' ID N05, SEQ ID N06, SEQ ID N07, SEQ ID N08, SEQ ID N09, SEQ ID N039, SEQ ID N042 and SEQ ID N043, complementary sequences thereof and equivalent sequences thereof, in 3 0 3 6 9 4 particular the nucleotide sequences having, for any sequence of 100 contiguous monomers, at least 50% and preferably at least 70%. homology with a nucleotide sequence chosen from SEQ ID NOl, SEQ ID N02, SEQ XD N03, 5 SEQ ID N04, SEQ ID N05, SEQ ID N06, SEQ ID N07, SEQ ID N08, SEQ ID N09, SEQ ID N039, SEQ ID N042 and SEQ ID N043 and complementary sequences thereof, in order to obtain a diagnostic, prophylactic or therapeutic composition for detecting, preventing or treating an 10 infection by the said viral, material or a reactivation of the said viral material, associated with rheumatoid arthritis, iv) the use of a retroviral material, the pol gene of whose genome comprises an equivalent nucleotide sequence, and especially having at least 50% homology, preferably at least 65% homology, with a nucleotide sequence belonging to the pol gene of the genome of the retrovirus ERV-9 or HSERV-9, in order to obtain a diagnostic, prophylactic or therapeutic composition for 20 detecting, preventing or treating sua infection by the said viral material or a reactivation of the said viral material, associated with rheumatoid arthritis, v) the use of a retroviral material, the pol gene of whose genome codes for a peptide sequence having at least\ 50% and preferably at least 7.0% homology, with a \ peptid^ sequence coded for by the pol gene of the genome of the retrovirus ERV-9 or HSERV-9, in order to obtain a diagnostic, prophylactic or therapeutic composition for detecting,^ preventing or treating an infection by the 30 said viral, material or a reactivation of the said viral material, aVssociated with rheumatoid arthritis, vi) * the use of a retroviral material, the pol gene of whose genome codes for a peptide sequence having, for any contiguous sequence of at least 30 amino acids, at least 50% and preferably at least 70% homology with a peptide sequence coded for by a nucleotide sequence chosen from SEQ ID NOl, SEQ ID N02, SEQ ID N03, SEQ ID N04, SEQ ID N05, SEQ ID N06, SEQ ID N07, SEQ XD N08, SEQ XD N09, SEQ ID N039, SEQ ID N042 and SEQ ID N043 and 3 0 3 6 9 4 complementary sequences thereof, in order to obtain a diagnostic, prophylactic or therapeutic composition for detecting, preventing or treating an infection by the said viral material or a reactivation of the said viral 5 material, associated with rheumatoid arthritis, vii) the use of a nucleotide fragment whose nucleotide sequence comprises a nucleotide sequence chosen from SEQ XD NOl, SEQ ID N02, SEQ XD N03, SEQ XD N04, SEQ XD N05, SEQ ID N06, SEQ XD N07, SEQ XD N08, SEQ XD N09, SEQ XD N039, SEQ XD N042 and SEQ XD N043, complementary sequences thereof and equivalent sequences thereof, in particular the nucleotide sequences having, for any continuous sequence of 100 contiguous monomers, at least 50% and preferably at least 15 70% homology with a sequence chosen from SEQ ID NOl, SEQ XD N02, SEQ XD N03, SEQ XD N04, SEQ XD N05, SEQ XD N06, SEQ XD N07, SEQ XD N08, SEQ XD N09, SEQ XD N039, SEQ XD N042 and SEQ ID N043 and complementary sequences thereof* in order to obtain a diag-20 nostic, prophylactic or therapeutic composition for detecting, preventing or treating an infection by the said viral material or a reactivation of the said viral material, associated with rheumatoid arthritis, viii) the use of a specific primer comprising a 25 nucleotide sequence identical or equivalent to at least a part of the nucleotide sequence of a fragment defined in vii), in particular a nucleotide sequence having, for any sequence of 10 contiguous monomers, at least 70% homology with at least a part of the said fragment, in 30 order to amplify by polymerization an RNA or a DNA of a viral material associated with rheumatoid arthritis; according to a preferable use, the primer comprises a nucleotide sequence chosen from SEQ ID N016, SEQ XD N017, SEQ ID NOl8, SEQ ID NOl9, SEQ ID N020, SEQ ID N021, 35 SEQ ID N022, SEQ XD N023, SEQ XD N024, SEQ XD N025, SEQ XD N026, SEQ XD N031, SEQ XD N032, SEQ ID N033, SEQ ID N047, SEQ ID N048 and SEQ ID N049 and complementary sequences thereof, ix, the use of a probe comprising a nucleotide 3 0 3 6 ^ 4 sequence identical or equivalent to at: least a part o£ the nucleotide sequence o£ a fragment defined in vii) , in particular a nucleotide sequence having, for any sequence of 10 contiguous monomers, at least 70% homology with at 5 least a part of the said fragment, in order to obtain a composition to detect, separate or identify, in a biological sample, a viral material or a reactivation of the said viral .material, associated with rheumatoid arthritis; according to a preferable use, the probe comprises 10 a nucleotide sequences chosen from SEQ ID N03, SEQ ID N04, SEQ ID N05, SEQ ID N06, SEQ ID N07, SEQ XD N016, SEQ ID N017, SEQ ID N018, SEQ ID N019, SEQ ID N020, SEQ ID N021, SEQ ID N022, SEQ ID N023, SEQ ID N024, SEQ ID N025, SEQ ID N026, SEQ ID N031, SEQ XD tt032, 15 SEQ ID N033, SEQ ID N047, SEQ ID N048 and SEQ ID N049 and complementary sequences thereof, x) the use of a pathogenic and/or infective agent, in the purified or isolated state, different from the viral material defined in any one of points i) to vi), as derived from a viral strain chosen from the strains referred to respectively as POL-2, filed on 22.07.1992 with the ECACC under the access number V92072202, and MS7P6 filed on 08.01.93 with the ECACC under the access number V93010816, and from the variant 25 strains thereof, consisting of pathogenic and/or infective agents comprising at least one antigen which is recognized by at least one antibody directed against at least one antigen corresponding to either of the pathogenic emd/or infective agents of the abovementioned 3 0 viral strains POL-2 and HS7PG, which are respectively different from either retroviral material of the said strains, in order to obtain a diagnostic, prophylactic or therapeutic composition for detecting, preventing or treating an infection by the said pathogenic and/or 35 infective agent or a reactivation of the said pathogenic and/or infective agent, associated with rheumatoid arthritis, xi) the use of a pathogenic and/or infective agent, in the purified or isolated state, different from 3 b y 4 the viral material defined in any one of points i) to vi) , as produced by a cell line chosen from the lines referred to respectively as PLI-2 filed on 22.07.1992 with the ECACC under the access number 92072201, and 5 LM7PC filed on 08.01.93 with the ECACC under the access number 93010817, and by any infected cell cultures capable of producing at least either of the pathogenic and/or infective agents, and/or variants thereof, or by any infected cell culture capable of producing a 10 pathogenic and/or infective agent comprising at least one antigen which is recognized by at least one antibody directed against at least one antigen corresponding to either of the pathogenic and/or infective agents produced by the abovementioned lines PLI-2 and LM7PC, in order to 15 obtain a diagnostic, prophylactic or therapeutic composition for detecting, preventing or treating an infection by the said pathogenic and/or infective agent or a reactivation of the said pathogenic and/or infective agent, associated with rheumatoid arthritis, 20 xii) the use of a pathogenic and/or infective agent comprising a nucleic acid comprising a nucleotide sequence chosen from SEQ ID N010, SEQ ID NOll, SEQ ID N012, SEQ ID N040 and SEQ ID N041, complementary sequences thereof and equivalent sequences thereof, in 25 particular the nucleotide sequences having at least 70% and preferably at least 90% homology with a nucleotide sequence comprising a sequence chosen from SEQ ID N010, SEQ ID NOll, SEQ ID N012, SEQ ID N040 and SEQ ID N041 and complementary sequences thereof, in order to obtain a 30 diagnostic, prophylactic or therapeutic composition for detecting, preventing or treating an infection by the said pathogenic and/or infective agent or a reactivation of the said pathogenic and/or infective agent, associated with rheumatoid arthritis, xiii) the use of a nucleotide fragment comprising a nucleotide sequence chosen from SEQ ID N010, SEQ ID NOll, SEQ ID N012, SEQ ID N040 and SEQ ID N041, complementary sequences thereof and equivalent sequences thereof, in particular the nucleotide sequences having, for amy 8 - 3 6 94 sequence of 100 contiguous monomers, at least 70% and preferably at least 90% homology with a sequence chosen from SEQ ID N010, SEQ ID NOll, SEQ ID N012, SEQ ID N040 and SEQ ID N041 and complementary sequences thereof, in 5 order to obtain a diagnostic, prophylactic or therapeutic composition for detecting, preventing or treating an infection by the said pathogenic and/or infective agent or a reactivation of the said pathogenic and/or infective agent, associated with rheumatoid arthritis, 10 xiv) the use of a primer comprising a nucleotide sequence identical or equivalent to at least a part bf the nucleotide sequence of a fragment defined in xiii), in particular a nucleotide sequence having, for any sequence or 10 contiguous monomers, at least 90% homology 15 with at least a part of the said fragment, in order to amplify by polymerization an RNA or a DNA of a pathogenic and/or infective agent associated with rheumatoid arthritis; according to an advantageous use, the primer comprises a nucleotide sequence chosen from SEQ ID N013, 20 SEQ ID N014, SEQ ID N015, SEQ ID N027, SEQ ID N028, SEQ ID N029, SEQ ID N030, SEQ XD N034, SEQ ID N035, SEQ ID N036, SEQ ID N037, SEQ ID N044, SEQ ID N045 and SEQ ID N046 and complementary sequences thereof, xv) the use of a probe comprising a nucleotide 25 sequence identical or equivalent to at least a part of the nucleotide sequence of a fragment defined in xiii), in particular a nucleotide sequence having, for any sequence of 10 contiguous monomers, at least 90% homology with at least a part of the said fragment, in order to 30 obtain a diagnostic, prophylactic or therapeutic composition for detecting, preventing or treating an infection by the said pathogenic and/or infective agent or a reactivation of the said pathogenic and/or infective agent, associated with rheumatoid arthritis; according to 35 an advantageous use, the probe comprises a nucleotide sequence chosen from' SEQ ID N010, SEQ ID NOll, SEQ ID N013, SEQ ID N014, SEQ ID N015, SEQ ID N027, SEQ ID N028, SEQ ID N029, SEQ ID N030, SEQ ID N034, SEQ ID N035, SEQ ID N036, SEQ ID N037, SEQ ID N044, SEQ ID N045 and 3 0 3 6 9 4 SEQ ID N046 and complementary sequences thereof, xvi) the use of an association comprising two pathogenic and/or infective agents, in the isolated or purified state, 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 the said viruB, and a second agent, or a variant of the said second agent, these two pathogenic and/or infective agents being as those derived 10 from the same viral strain chosen from the strains referred to respectively as POL-2, filed on 22.07.1992 with the ECACC under the access number V92072202, and MS7PG filed on 08.01.93 with the ECACC under the access number V93010816, and from the variant strains thereof, 15 in order to obtain a diagnostic, prophylactic or therapeutic composition for detecting, preventing or treating an infection by a first pathogenic emd/or infective agent, and a second pathogenic and/or infective agent, associated with rheumatoid arthritis, or a reactivation 20 of either the said first agent or the said second agent, xvii) the use of an association comprising two pathogenic and/or infective agents, in the isolated or purified state, namely, a first agent consisting of a human virus possessing reverse transcriptase activity, and related to a family of endogenous retroviral elements, or a variant of the said virus, and a second agent, or a variant of the said second agent, these two pathogenic and/or infective agents being as those produced by the same cell line chosen from the lines 30 referred to respectively as PLI-2, filed on 22.07.1992 with the ECACC under the access number 92072201, and LM7PC, filed on 08.01.93 with the ECACC under the access number 93010817, and by any infected cell cultures capable of producing at least either of the pathogenic 35 and/or infective agents, and/or variants thereof, in order to obtain a diagnostic, prophylactic or therapeutic composition for detecting, preventing or treating em infection by a first pathogenic and/or infective agent, and a second pathogenic emd/or infective agent, associa- Tli A * - 10 6G s 4 ted with rheumatoid arthritis, or a reactivation of either of the said first agent and the said second agent, xviii) the use of an association comprising two pathogenic and/or infective agents, in the isolated or 5 purified state, namely a first agent consisting of a virus, or of a variant of the said virus, the genome of which comprises a nucleotide sequence chosen from SEQ ID NOl, SEQ ID N02, SEQ ID N03, SEQ ID N04, SEQ ID N05, SEQ ID N06, SEQ ID N07, SEQ ID N08, SEQ ID N09, 10 SEQ ID N039, SEQ ID N042 and SEQ ID N043, complementary sequences thereof and equivalent sequences thereof, in particular the nucleotide sequences having, for any sequence of 100 contiguous monomers, at least 50% and preferably at least 70% homology with a nucleotide 15 sequence chosen from SEQ ID NOl, SEQ ID N02, SEQ ID N03, SEQ ID N04, SEQ ID N05, SEQ ID NO6, SEQ ID N07, SEQ ID N08, SEQ ID N09, SEQ ID N039, SEQ ID N042 and SEQ ID N043 and complementary sequences thereof, and a second pathogenic and/or infective agent, the genome of 20 which comprises a nucleotide sequence chosen from SEQ ID N010, SEQ ID NOll, and SEQ ID N012, SEQ ID N040, SEQ ID N041 and complementary sequences thereof and equivalent sequences thereof, in particular the nucleotide sequences having, for any sequence of 100 25 contiguous monomers, at least 70% and preferably at least 90% homology with a nucleotide sequence chosen from SEQ ID N010, SEQ ID NOll, SEQ ID N012, SEQ ID N040 and SEQ ID N041 and complementary sequences thereof, in order to obtain a diagnostic, prophylactic or therapeutic 30 composition for detecting, preventing or treating an infection by a first pathogenic and/or infective agent, and a second pathogenic and/or infective agent, associated with rheumatoid arthritis, or a reactivation of either the said first agent or the said second agent, 35 xix) the use of an association of nucleotide fragments comprising a first fragment' whose nucleotide sequence comprises a nucleotide sequence chosen from SEQ ID NOl, SEQ ID N02, SEQ ID N03, SEQ ID N04, SEQ ID N05, SEQ ID NO6, SEQ ID N07, SEQ ID N08, SEQ ID N09, 3 0 h 11 - SEQ ID N039, SEQ ID N042 and SEQ ID N043, complementary sequences thereof and equivalent sequences thereof, in particular the nucleotide sequences having, for any sequence of 100 contiguous monomers, at least 50% and 5 preferably at least 70% homology with a nucleotide sequence chosen from SEQ ID NOl, SEQ ID N02, SEQ ID N03, SEQ ID N04, SEQ ID N05, SEQ ID N06, SEQ ID N07, SEQ ID N08, SEQ ID N09, SEQ ID N039, SEQ ID N042 and SEQ ID N043 and complementary sequences thereof, and a 10 second fragment whose nucleotide sequence comprises a nucleotide sequence chosen from SEQ ID NOlO, SEQ ID NOll, SEQ ID N012, SEQ ID N040 and SEQ ID N041, complementary sequences thereof and equivalent sequences thereof, in particular the nucleotide sequences having, for' any 15 sequence of 100 contiguous monomers, at least 70% and preferably at least 90% homology with a nucleotide sequence chosen from SEQ ID NOlO, SEQ ID NOll, SEQ ID N012, SEQ ID N040 and . SEQ XD N041 and complementary sequences thereof, each of the said fragments being in 20 particular a probe, in order to obtain a diagnostic, prophylactic or therapeutic composition for detecting a first pathogenic and/or infective agent, and a second pathogenic and/or infective agent, associated with rheumatoid arthritis, or a reactivation of either the 25 said first agent or the said second agent, xx) the use of an association comprising a first polypeptide partially or totally coded for by the first nucleotide fragment defined in ix) , and a second polypeptide partially or totally coded for by the second 30 nucleotide fragment defined in ix) , in order to obtain a diagnostic, prophylactic or therapeutic composition for detecting a first pathogenic and/or infective agent, and a second pathogenic and/or infective agent, associated with rheumatoid arthritis; according to an advantageous 35 use, this association comprises a first ligand, in particular an antibody, which is specific for the first polypeptide, and a second ligand, in particular an antibody, which is specific for the second polypeptide, the said first and second polypeptides being defined 3 0 o c 4 above, xxi) a process for the production of a first pathogenic and/or infective agent defined in any one of points i) to vi) and/or of a second pathogenic and/or infective agent defined in any one of points x) to xii), associated with rheumatoid polyarthritis, characterized in that cells from synovial fluid punctures are cultured in vitro, these cells being chosen in particular from the desquamated fibroblasts and synoviocytes of articular 10 fluid, from patients suffering from rheumatoid arthritis, xxii) a process for the production of a first pathogenic and/or infective agent defined in any one of points i) to vi) and/or of a second pathogenic and/or infective agent defined in any one of points x) to xii) , associated with rheumatoid arthritis, characterized in that B lymphocyte cells immortalized by Epstein-Barr virus are cultured in vitro, these cells coining from patients suffering from rheumatoid arthritis, xxiii) a nucleotide fragment whose nucleotide 20 sequence comprises a sequence chosen from SEQ ID N039, SEQ ID N042, SEQ ID N043, and complementary sequences thereof and equivalent sequences thereof, in particular the nucleotide sequences having, for any sequence of 100 contiguous monomers, at least 50% and preferably at least 25 70% homology with a sequence chosen from SEQ ID N039, SEQ ID N042 and SEQ ID N043 and complementary sequences thereof, xxiv) a specific primer for the amplification by polymerization of an KK& or a DNA of a viral material defined in any one of points i) ii) ill) iv) v) or vi), comprising a nucleotide sequence identical or equivalent to at least a part of the nucleotide sequence of a fragment according to xxiii), in particular a nucleotide sequence having, for any sequence of 10 contiguous mono-35 mers, at least 70% homology with at least a part of the said fragment; in particular, this primer comprises a nucleotide sequence chosen from SEQ ID N047, SEQ ID N048 and SEQ ID N049 and complementary sequences thereof, xxv) a probe capable of specifically hybridizing '30 3 6 t with an RNA or a DNA of a viral material defined in any one of points i) ii) iii) iv) v) or vi), comprising a nucleotide sequence identical or equivalent to at least a part of the nucleotide sequence of a fragment according 5 to xxiii), in particular a nucleotide sequence having, for any sequence of 10 contiguous monomers, at least 70% homology with at least a part of the said fragment; in particular, this probe comprises a nucleotide sequence chosen from SEQ ID N047, SEQ ID N048 and SEQ ID N049 and 10 complementary sequences thereof, xxvi) a nucleotide fragment whose nucleotide sequence comprises a sequence chosen from SEQ ID N040 and SEQ ID N041, complementary sequences thereof and equivalent sequences thereof, in particular the nucleotide sequences having, for any sequence of 100 contiguous monomers, at least 70% and preferably at least 90% homology with a sequence chosen from SEQ ID N040 and SEQ ID N041 and complementary sequences thereof, xxvii) a specific primer for the amplification by 20 polymerization of an SNA or a DNA of a pathogenic and/or infective agent defined in any one of points x) xi) or xii) , comprising a nucleotide sequence identical or equivalent to at least a part of the nucleotide sequence of a fragment according to xxvi), in particular a 25 nucleotide sequence having, for any sequence of 10 contiguous monomers, at least 90% homology with at least a part of the said fragment; in particular, this primer comprises a nucleotide sequence chosen from SEQ ID N044, SEQ ID N045 and SEQ ID N046 and complementary sequences 3 0 thereof, xxviii) a probe capable of specifically hybridizing with an RNA or a DNA of a viral material defined in any one of points x) ix) xi) or xii), comprising a nucleotide sequence identical or equivalent to at least a part of the nucleotide sequence of a fragment according to xxvi), in particular a nucleotide sequence having, for any sequence of 10 contiguous monomers, at least 90% homology with at least a part of the said fragment; in particular, this probe comprises a nucleotide sequence 3 0 3 6 9 4 chosen from SEQ ID N044, SEQ ID N045 and SEQ ID N046 and complementary sequences thereof.

Before going into the invention in detail, various terms used in the description and claims are now 5 defined: - strain or isolate is understood to refer to any infective and/ox pathogenic biological fraction containing, for example, viruses and/or bacteria emd/or parasites, and generating a pathogenic and/or antigenic capacity, harboured by a culture or a living host; by way of example, a viral strain according to the above definition may contain a co-infective agent, for example a pathogenic protist, - the teem "MSRV" used in the present description 15 denotes any pathogenic and/or infective agent, associated with MS or with RA, in particular a viral species, attenuated strains of the said viral species or defective interfering particles derived from this species. It is known that viruses and particularly viruses containing 20 RNA have a variability, in particular following relatively high levels of spontaneous mutation (9), which will be taken into account below to define the notion of equivalence, - the expression human virus refers to a virus 25 capable of infecting human beings, - taking into account all the natural or iaduced variations which may be encountered when carrying out the present invention, the subjects of this invention, defined above and in the claims, have been expressed comprising therein the equivalents or derivatives of the various biological materials defined below, in particular of the nucleotide or peptide homologous sequences, - the variant of a virus or of a pathogenic and/or infective agent according to the invention com- prises at least one antigen recognized by at least one antibody directed against at least one antigen corresponding to the said virus and/or of the said pathogenic emd/or infective agent, and/or a genome in which any part is detected by at least one hybridization 3 0 3 6 n probe, and/or at least one nucleotide amplification primer specific for the said virus and/or pathogenic and/or infective agent, for example such as those having a nucleotide sequence chosen from SEQ ID N013 to SEQ ID 5 N038 and SEQ ID N044 to SEQ ID N049 and complementary sequences thereof, under specific hybridization conditions which are well known to those skilled in the art, according to the invention, a nucleotide 10 fragment or an oligonucleotide or a polynucleotide is an arrangement of monomers, or a biopolymer, characterized by the informational sequence of the natural nucleic acids, which are capable of hybridizing with amy other nucleotide fragment under predetermined conditions, it 15 being possible for the arrangement to contain monomers of different chemical structures and to be obtained from a natural nucleic acid molecule and/or by genetic recombination and/or by.chemical synthesis, - thus, a monomer may be a natural nucleotide of 2 0 nucleic acid, the constituent elements of which are a sugar, a phosphate group and a nitrogenous base; in RNA the sugar is ribose, in DNA the sugar is 2-deoxyribose; depending on whether the nucleic acid is DNA or RNA, the nitrogenous base is chosen from adenine, guanine, uracil, 25 cytosine and thymine; or the nucleotide may be modified in at least one of the three constituent elements; by way of example, the modification may occur in the bases, generating modified bases such as inosine, 5-methyl-deoxycytidine, deoxyuridine, 5- (dimethylamino) deoxy-30 uridine, 2,6-diaminopurine, 5-bromodeoxyuridine and any other modified base which promotes hybridization; aB regards the sugar, the modification may consist of the replacement of at least one deoxyribose by a polyamide (10), and as regards the phosphate group, the modifica-35 tion may consist of its replacement by esters, chosen in particular from diphosphate, alkyl and aryl phosphonate and phosphorothioate esters, - the expression "information sequence" refers to any ordered sequence of monomers, of which the chemical 3 6 8 nature and the order in a reference direction do or do not constitute an item of functional information of the same quality as that of the natural nucleic acids, - the expression hybridization refers to the 5 process during which, under suitable operating conditions, two nucleotide fragments having sufficiently complementary sequences pair together to form a multiple strand, in particular a double or triple strand, preferably in the form of a helix, - a probe comprises a nucleotide fragment syn thesized chemically or obtained by digestion or enzymatic cleavage of a longer nucleotide fragment, comprising at least six monomers, advantageously from 10 to 100 mono- . mers, preferably 10 to 30 monomers, and possessing 15 hybridization specificity under specific conditions; preferably, a probe possessing less than 10 monomers is not used alone, but is used in the presence of other probes which are of the same small size or otherwise; under certain specific conditions, it may be useful to 20 use longer probes, for example of more than 100 monomers in size; a probe may be used in particular for diagnostic purposes and such probes will be, for example, capture and/or detection probes, - the capture probe may be immobilized on a solid 25 support by any suitable means, that is to say directly or indirectly, for example by covalent bonding or passive adsorption, - the detection probe may be labelled by means of a label chosen in particular from radioactive isotopes, 3 0 enzymes chosen in particular from peroxidase and alkaline phosphatase and those capable of hydrolysing a chromo-genic, fluorogenic or luminescent substrate, chromophoric chemical compounds, chromogenic, fluorogenic or luminescent compounds, analogues of nucleotide bases, and 35 biotin, - the probes used for diagnostic purposes of the invention may be used in all the known hybridization techniques and in particular the techniques known as "DOT-BLOT" (11), "SOUTHERN BLOT" (12), "NORTHERN BLOT", 36 9 4 which is a technique identical to the "SOUTHERN BLOT" technique but which uses RNA as target, and the SANDWICH technique (13); advantageously the SANDWICH technique is used in the present invention, comprising a specific 5 capture probe and/or a specific detection probe, it being understood that the capture probe and the detection probe must have a nucleotide sequence which is at least partially different, - the invention also covers a probe capable of 10 hybridizing in vivo or in vitro with RNA and/or DNA, in order to block the phenomena of replication, in particular translation and/or transcription, and/or to degrade the said DNA and/or RNA, - a prixaer is a probe comprising . at least six 15 monomers, and advantageously from 10 to 30 monomers, possessing specificity of hybridization under specific conditions, in order to initiate an enzymatic polymerization, for example in. an amplification technique such as PCR (Polymerase Chain Reaction), in an elongation 20 process such as sequencing, in a reverse transcription method or the like, - two nucleotide or peptide sequences are said to be equivalent or derived with respect to each other, or with respect to a reference sequence, if, functionally, the corresponding biopolymers may perform substantially the same role, without being identical, with regard to the application or use considered, or in the technique in which they are involved; sequences which are in particular equivalent are two sequences obtained on the basis of 30 the natural variability, in particular spontaneous mutation of the species from which they have been identified, or induced, as well as homologous sequences, homology being defined below, - the term variability refers to any spontaneous 35 or induced modification of a sequence, in particular by substitution and/or insertion emd/or deletion of nucleotides and/or nucleotide fragments, emd/or extension emd/or shortening of the sequence at at least one of the ends; an unnatural varis&ility may result from the 3 0 3 6 9 genetic engineering techniques used, for example from the choice of the degenerate or non-degenerate synthesis primers selected in order to amplify a nucleic acid; thiB variability may be reflected by modifications of any 5 starting sequence, considered as reference, and which may be expressed by a degree of homology relative to the said reference sequence, the homology characterizes the degree of similarity of two compared nucleotide or peptide frag-10 ments; it is measured by the percentage of similarity, which is determined in particular by direct comparison of nucleotide or peptide sequences, relative to reference nucleotide or peptide sequences, - this percentage of similarity has been specifi-15 cally determined for the nucleotide fragments forming part of the present invention, which are homologous with the fragments identified by SEQ ID NOl to SEQ ID N09, SEQ ID N039, SEQ ID N042 and SEQ ID N043, (MSRV-1) on the one hand, and those which are homologous with the frag-20 ments identified by SEQ ID NOlO to SEQ ID N012 and SEQ ID N040 to SEQ ID N043 (MSRV-2), on the other hand, as well as for the probes and primers homologous with the probes and primers identified by SEQ ID N016 to SEQ ID N026, SEQ ID N031 to SEQ ID N033 and SEQ ID N047 25 to SEQ ID N049 on the one hand, and to the probes and primers identified by SEQ ID N013 to SEQ ID N015, SEQ ID N027 to SEQ ID N030, SEQ ID N034 to SEQ ID N037, and SEQ ID N044 to SEQ ID N046, on the other hand; by way of example, the lowest percentage of similarity observed 30 between the various general consensus sequences of nucleic acids obtained from viral RNA fragments of MSRV-1, derived from LM7PC and PLI-2 lines according to a procedure outlined later, is 67% in the region described in Figure 2. 3 5 - any nucleotide fragment is said to be equival ent to or derived from a reference fragment if it has a nucleotide sequence equivalent to the reference sequence; according to the above definition, the following are, in particular, equivalent to a reference nucleotide frag- 3 6 merit: a) any fragment capable of hybridizing at least partially with the complement of the reference fragment b) any fragment whose alignment with the reference fragment leads to the demonstration of identical contiguous bases, in greater numbers than with any other fragment originating from another taxonomic group c) any fragment resulting or liable to result from the natural variability of the species from which it is obtained d) any fragment liable to result from genetic engineering techniques applied to the reference fragment e) any fragment, containing at least eight contiguous nucleotides, coding for a peptide which is homologous with or identical to the peptide coded for by the reference fragment f) any fragment differing from the reference fragment by insertion, deletion, substitution of at least one monomer, extension or shortening at at least one of its ends; for example, any fragment corresponding to the reference fragment flanked at at least one of its ends by a nucleotide sequence which does not code for a polypeptide, - the term polypeptide refers in particular to any peptide of at least two amino acids, in particular oligopeptide or protein, extracted, separated or substantially isolated or synthesized through human intervention, in particular those obtained by chemical synthesis or by expression in a recombinant organism - the expression polypeptide which is at least partly coded for by a nucleotide fragment is understood to refer to a polypeptide having at least 3 amino acids coded for by at least 9 contiguous monomers included in the said nucleotide fragment, - an amino acid is said to be analogous to smother amino acid when their respective physicochemical characteristics, such as polarity, hydrophobicity and/or basicity and/or acidity and/or neutrality, are substantially the same; thus, a leucine is analogous to an 3 0 3 6 9 4 isoleucine. - any polypeptide is said to be equivalent to or derived from a reference polypeptide if the polypeptides compared have substantially the same properties, and in 5 particular the same antigenic, immunological, enzymo-logical and/or molecular recognition properties; the following are, in particular, equivalent to a reference polypeptide: a) any polypeptide possessing a sequence in which 10 at least one amino acid has been substituted with an analogous amino acid, b) any polypeptide having em equivalent peptide sequence, obtained by natural or induced variation of the said reference polypeptide and/or of the nucleotide fragment coding for the said polypeptide, c) a mimotope of the said reference polypeptide, d) any polypeptide in whose sequence one or more amino acids from the L series are replaced by an amino acid from the D series, and vice versa, e) any polypeptide in which at least one of the CO-NH linkages, and advantageously all of the CO-NH linkages, of the peptide chain of the corresponding parent peptide (not containing any NH-CO linkages in its peptide chain) is (are) replaced by one (or more) NH-CO 25 linkage(s), f) any polypeptide in which at least one of the CO-NH linkages, and advantageously all of the CO-NH linkages, of the peptide chain of the corresponding parent peptide (not containing any NH-CO linkages in its peptide chain) is (are) replaced by one (or more) NH-CO linkage(s), the chirality of each aminoacyl residue, whether or not this is involved in one or more above-mentioned CO-NH linkages, being either conserved or inverted relative to the corresponding aminoacyl residues 35 constituting the said parent peptide, these compounds of the peptide type also being denoted as immunoretroids, g) any polypeptide in whose sequence a modification of the side chains of the amino acids has been introduced, for example such as an acetylation of the 3 0 3 6 0 amine functions, a carboxylation of the thiol functions or an esterification of the carboxylic functions, h) any polypeptide in whose sequence one or more peptide linkages have been modified, such as, for example, the carba, retro, inverso, retro-inverso, reduced and methyleneoxy linkages. i) any polypeptide of which at least one antigen is recognized by an antigen of the reference polypeptide, - the percentage of similarity characterizing the 10 homology of two compared peptide fragments is, according to the present invention, at least 50% and preferably at least 70%.

Given that a virus possessing reverse transcriptase enzymatic activity may be genetically characterized 15 both in RNA and in DMA form, mention will be made both of the viral DNA and the viral RMA in order to characterize the sequences relating to a virus possessing such reverse transcriptase activity (MSRV-1).

Given that the pathogenic and/or infective agent 20 (MSRV-2) has been detected both as DNA and as RNA in the infected cells, it may also be characterized in DNA or RNA form.

The expressions of order used in the present description and the claims, such as "first nucleotide 25 sequence" are not adopted to express a particular order, but to define the invention more clearly.

A better understanding of the invention will be gained on reading the detailed description which follows, made with reference to the attached figures in which: 30 - Figure 1 represents the sequence of MSRV-2A type obtained from LM7 cultures according to Shih's procedure (8); this sequence is identified under the reference SEQ ID NOlO, - Figure 2 represents general nucleic acid con-35 sensus sequences of the MSRV-1B sequences amplified by the PCR technique in the "pol" region, using viral D&A derived from the LM7PC and PLI-2 lines, identified under the references SEQ ID N03, SEQ ID N04, SEQ ID N05 and SEQ ID N06, and the common consensus with amplification 3 0 5 68 4 primers bearing the reference SEQ ID N07f - Figure 3 represents the phylogenetic tree of the sequences of MSRV-1B type obtained by PCR in the "pol" region defined by Shih (8), - Figure 4 gives the definition of a functional reading frame for each family of MSRV-1B/"PCR pol" type, the said families A to D being defined respectively by the nucleotide sequences SEQ ID N03, SEQ ID N04, SEQ ID N05 and SEQ ID N06 described in Figure 2, 10 - Figure 5 gives an example of consensus of the MSRV-2B sequences, identified by SEQ ID NOll, - Figure 6 represents the nucleotide sequence of the clone PSJ17 (SEQ ID N09) , - Figure 7 represents the nucleotide sequence 15 SEQ ID N08, of the clone referred to as M003-P004, - Figure 8 represents the nucleotide sequence SEQ ID N02 of the clone Fll-1; the part referenced between two arrows in the region of the primer corresponds to a variability imposed by the choice of primer which served to clone Fll-1; the translation into amino acids is represented in this same figure, - Figure 9 represents the nucleotide sequence SEQ ID NOl, and a functional frame for possible reading of SEQ ID NOl into amino acids; the consensus sequences of the retroviral reverse transcriptases are underlined in this sequence, - Figure 10 represents the nucleotide sequence SEQ ID NOl2 of the clone referred to as MSRV2EL1, - Figure 11, consisting of three successive 30 plates 11/23 to 13/23, represents the translation of SEQ ID N012, including the primer SEQ ID N013, into amino acids, according to 6 possible reading frames, - Figure 12 shows an alignment of the sequence MSRV2-A (SEQ ID NOlO) on the sequence MSRV2-EL1 (SEQ ID N012); the region of hybridization of the primer 1 identified under the reference SEQ ID N013 (excluding the cloning tail) is boxed in this same representation; that of the primer identified under the reference SEQ ID N014 is indicated between square brackets, 3 0 3 6 9 4 - Figure 13 represents the measurement of the reverse transcriptase activity under the conditions defined in Example 7, in the fractions of a sucrose gradient on which a sedimentation has been performed to the equilibrium point of infective emd/or pathogenic agents present in the supernatant of cultures of cells from articular fluids of rheumatoid arthritis, as is described in Example 7; the curve represents the variations in reverse transcriptase activity in the various 10 fractions of the gradient; this activity is measured in DPM (disintegrations per minute) on the y-axis; the x-axis represents the fractions collected on the gradient by order of increasing density (fractions 1 to 10), - Figure 14, divided into Figures 14A, 14S and 15 14C, shows, in Figure 14A, the sequence of the clone "MSRV-lpolPR" identified by SEQ ZD N039, obtained on a bample of RA. under the conditions defined in Example 9; Figures 14B and 14C show the alignment of the sequence of this clone with SEQ ID NOl and SEQ ID N06 respectively, 20 corresponding to sequences of the retrovirus MSRV-1 obtained in samples from an MS source, - Figure 15, divided into Figures 15A, 15B and 15C, shows, in Figure ISA, the sequence of the clone "MSRV2sPR" identified by SEQ ID N040, obtained on a sample of RA under the conditions defined in Example 9; Figures 15B and 15C show the alignment of the sequence of this clone with SEQ ID NOlO and SEQ ID N012 respectively, corresponding to sequences of the infective agent MSRV-2 obtained in samples from an MS source, 30 - Figure 16 shows the sequence of the clone MSRV2cPR identified by SEQ ID N041, obtained using cells from an articular fluid of RA, under the conditions defined in Example 10, - Figure 17 shows the sequence of the clone 35 MSRV2cPR identified by SEQ ID N041, obtained using cells from an articular fluid of RA, and the alignment of the terminal part (654-705) of the sequence of this clone with SEQ ID NOip, corresponding to a sequence of the infective agent MSRV-2 obtained in samples from an MS 24 - 3 69 4 source, - Figure 18 shows the sequence of the clone MSRV2cPR identified by SEQ XD N041, obtained using cells from an articular fluid of RA, and the alignment of the sequence of this clone with SEQ ID N012, corresponding to a sequence of the infective agent MSRV-2 obtained in samples from an MS source, - Figure 19 shows the sequence of the clone MSRVlnPR identified by SEQ ID N042, obtained using an articular fluid from RA, under the conditions defined in Example 10, - Figure 20 shows the sequence of the clone MSRVlnPR identified by SEQ XD N043, obtained using cells from an articular fluid of RA, and the alignment of the sequence of this clone with SEQ ID NOl, corresponding to a sequence of the retrovirus MSRV-1 obtained using samples from an MS source.

EXAMPLE 1: PRODUCTION OF MSRV-2 CLONES REFERRED TO AS MSRV-2A, BY AMPLIFICATION OF THE CONSERVED REGIONS 20 OF THE RNA-DEPENDENT DNA-POLYMERASE GENES, ON A PREPARATION OF INFECTIVE AGENT PURIFIED FROM A CULTURE OF CELLS FROM THE LM7 LINE The molecular approach consisted in using a PCR technique (8) which makes it possible to amplify a rela-25 tively conserved region of the pol gene of exogenous and endogenous retroviruses, but also of viruses coding for an enzyme with reverse transcriptase (RT) activity such as, in particular, hepatitis B virus ancl, implicitly, of any RNA-dependent DNA polymerase or enzyme gene, having 30 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 procedure (14) from the supernat&nts of the original LM7 35 culture (?) stored thereafter in a deep-freezer at -80°C. The fractions containing the peak of RT activity of LM7 type are taken up in one volume of a buffer containing guanidine thiocyanate (15) and are stored at -80°C until extraction of the nucleic acids according to the 3 5 9 4 technique described by P. Chomzynski (15).

Prior to the PCR reaction, the RNA of the sample was transcribed into complementary DNA (cDNA) with so-called "random" primers (mixture of hexanucleotides) using the "cDNA synthesis system plus" kit (Amersham), according to the manufacturer's instructions and based on 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 fxl of DNA solution were mixed with 5 /il of sterile distilled water, 1 /il of a 10-fold concentrated ligation buffer "10X LIGATION BUFFER", 2 fil of "pCR™ VECTOR" (25 ng/ml) and 1 /il of "TA DNA LIGASE". This mixture was incubated overnight at 12 °C. The following steps were performed in accordance with the instructions of the TA Cloning9 kit. At the end of the procedure, the white colonies of recombinant bacteria were picked out so as to be cultured and to allow the extraction of the plasmids incorporated according to the so-called "miniprep" procedure (16). The plasmid preparation from each recombinant colony was cut by a suitable restriction enzyme and analysed on agarose gel. The 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 promoter Sp6 present on the cloning plasmid of the "TA cloning kit". The reaction prior to the sequencing was then carried out according to the method recommended for the use of the sequencing kit "Prism ready reaction kit dye deoxy-terminator cycle sequencing kit" (Applied Biosystems, ref. 401384) and automatic sequencing was performed with the "Automatic Sequencer" model 373 A machine from Applied Biosystems, according to the manufacturer's ins truetions.

The sequences obtained were then analysed using Mac Vector® and Geneworks® software on a Genebank® computerized data bank for the nucleic acid sequences, and Swiss Prot® for the amino acid sequences deduced from the reading frames demonstrated in the nucleic acid sequences. Analysis of the sequences obtained from the viral sample originating from the thawed LM7 super-5 natants, and purified at the peak: of reverse transcriptase activity on a sucrose, gradient, revealed a majority . population of clones (about 42% of the clones), relative to the extent of individual representation of the other sequences (always less than 5%, or even 10% for a small 10 number of cases), and having partial homologies with known retroviruses in the expected "pol" region. This clone is referred to as MSRV2-A and is identified by SEQ ID NOlO (cf. Fig. 1). The region amplified between the PCR primers is homologous with the corresponding sequence 15 MSRV2-B identified by SEQ ID NOll (c£. Fig. 5), described in Example 2. The differences observed in the sequences located at the PCR primers are explained by the use of degenerate primers as a mixture, used under various technical conditions. Interrogation of the Genebank® data 20 bank has not hitherto made It possible to reveal an identical sequence or one having significant homologies.

This sequence is presented in Figure 1. The sequence presented in Figure 1 possesses an open reading frame in frame with the two PCR primers found at the 25 ends, but it is shorter than the set of retroviral sequences known in the expected region between these primers. A deletion of 45 base pairs (15 amino acids) is observed therein, relative to the corresponding retroviral sequences (8), following the sequence of the 30 upstream primer whereas the sequences preceding the downstream primer are present. However, the reading frame is open and uninterrupted throughout the sequence including the primers and the amino acid sequence deduced has significant homology with the corresponding region of the 35 known retroviruses. In the sequence lying inside the PCR primers, the amino acids Glu, Arg, Gin, Pro and Asp, which are normally fairly well conserved in this pol region of retroviruses and of known viruses with reverse transcriptase activity (8), are conserved at the right 3 0 3 6 9 4 positions in the reading frame of the novel sequence.

Lastly, given that this sequence is sufficiently divergent from the retroviral sequences already described in the data banks, the notion may be put forward that 5 this is a sequence belonging a novel infective and/or pathogenic agent, referred to as MSRV-2a. According to the analysis of the sequences obtained, this agent is related in principle to a retrovirus, but, given the technique used in order to obtain this sequence, it may 10 also be a virus containing DMA whose genome codes for an enzyme which incidentally possesses reverse transcriptaise activity, as is the case, for example, with the hepatitis B virus, HBV (8). Furthermore, the random nature of the degenerate primers used for this PCR 15 amplification technique may very well have allowed, on account of unforeseen sequence homologies or conserved sites in the gene of a related enzyme, the amplification of a nucleic acid originating from a prokaryotic or eukaryotic pathogenic and/or co-infective agent 20 (protist) .

EXAMPLE 2: PRODUCTION OF CLONES REFERRED TO AS MSRV-1B and MSRV-2B, RESPECTIVELY DEFINING A RETROVIRUS MSRV-1 AND CO-INFECTIVE AGENT MSRV2, BY "NESTED" PRC AMPLIFICATION OF TEE CONSERVED POL REGIONS OF RETRO-25 VIRUSES ON VIRION PREPARATIONS DERIVED FROM THE LM7PC AND PLI-2 LINES A PCR technique derived from the technique published by Shih (8) was used. This technique makes it possible, by treatment of all of the components of the 30 reaction medium with DNase to remove all trace of contaminating DNA. 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 35 from an amount of RNA which is small at the start and further reduced in the sample by the interfering action of the DNase on the RNA. Indeed, the DNase is used under conditions of excess activity which make it possible to remove all trace of contaminating DNA before inactivation 3036 94 - 28 of this enzyme remaining in the sample by heating at 85°C for 10 minutes. This variant of the PCR technique described by Shih (8) was used on a cDNA synthesized from the nucleic acids of fractions of infective particles 5 purified on a sucrose gradient according to the technique described by H. Perron (14) from the "POL-2n 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 10 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 92/20189.

After cloning, with the TA Cloning Kit®, of the 15 products amplified by this technique and analysis of the sequence using the automatic sequencer according to the description given in Example 1, the sequences were analysed using the Geneworks® software, on the latest available version of the Genebank® data bank. 20 The sequences cloned and sequenced from these samples correspond in particular to two types of sequence: a first type of sequence, found in the majority of the clones (55% of the clones derived from the POL-2 isolates of the PLI-2 culture and 67% of the clones 25 derived from the MS7PG isolates of the LM7PC cultures) which corresponds to a family of "pol" sequences similar to but different from the endogenous human retrovirus referred to as ERV-9 or HSERV-9, and a second type of sequence which corresponds to sequences which are very 30 highly homologous with the sequence attributed to an infective and/or pathogenic agent referred to above as MSRV-2.

The sequences of the first type representing the majority of the clones consist of sequences whose vari-35 ability makes it possible to define four sub-families of sequences. These sub-families are sufficiently similar to each other for them to be considered as quasi-species originating from the same retrovirus, as is well known for the HIV-1 retrovirus (17), or as a result of inter 3 0 35 94 ference with several endogenous proviruses co-regulated in the producing cells. These more or less defective endogenous elements are sensitive to the same regulatory signals which may be generated by a replicative provirus, 5 since they belong to the same family of endogenous retroviruses (18). This new family of endogenous retroviruses or, alternatively, this new retroviral species whose quasi-species generation was obtained in culture and which contains a consensus of the sequences described 10 below is referred to as MSRV-1B.

Figure 2 presents the general consensus sequences of the sequences of the various MSRV-1B clones sequenced during this experiment, these sequences being identified respectively as SEQ ID N03, SEQ ID N04, SEQ ID N05 and 15 SEQ ID N06. These sequences have a nucleic acid homology ranging from 70% to 88% with the HSERV9 sequence of reference X57147 and M37638 in the Genebank® data base. The phylogenetic tree of these sequences is presented in Figure 3. In this figure, the sub-families A, B, C and D 2 0 represent the sequences which have been predominantly found in similar experiments repeated subsequently, in the pure RNA samples of virions purified from the MS7PG and POL-2 isolates. From these families of sequences, four "consensus" nucleic acid sequences representative of 25 different quasi-species of a possibly exogenous MSRV-1B retrovirus, or different sub-families of an MSRV-1B endogenous retrovirus, were deifined. These representative consensus sequences are presented in Figure 4, with the translation into amino acids. A functional reading frame 3 0 exists for each sub-family of these MSRV-1B sequences, and it may be seen that the functional open reading frame corresponds in each case to the amino acid sequence on the second line under the nucleic acid sequence. The general consensus of the MSRV-1B sequence identified by 35 SEQ ID N07 and obtained by this PCR technique in the "pol" region is presented in Figure 2.

The second type of sequence representing the majority of the clones sequenced is represented by the MSRV-2B sequence present in Figure 5 and identified by 3 U o u » H SEQ ID NOll. The region amplified between the PCR primers is homologous, to the nearest base, with the MSRV2-A sequence (SEQ ID NOlO according to Fig. 1) lying inside the PCR primers which is described in Example 1. The differences observed in the sequences corresponding to the PCR primers are explained by the use of degenerate primers as a mixture used under various technical conditions .

The sequences MSRV-2 A (SEQ ID NOlO) and MSRV-2B (SEQ ID NOll) are, from the evidence, homologous, or even identical, derived from the same organism and sufficiently divergent from the retroviral sequences already described in the data banks to make it possible to suggest that this is a region of sequence belonging to a novel infective agent, referred to as MSRV-2. According to the analysis of the first sequences obtained, this infective agent would appear in principle to be related to a retrovirus, but, given the technique used to obtain this sequence, it may also be a DNA virus whose genome codes for an enzyme which incidentally possesses reverse transcriptase activity, as is the case, for exang>le, with the hepatitis B virus, HBV (8). Furthermore, the random nature of the degenerate primers used for this PCR amplification technique may very well have allowed, on account of unforeseen sequence homologies or of sites conserved in the gene of a related enzyme, the amplification of a nucleic acid originating from a prokaryotic or eukaryotic pathogenic and/or co-infective agent (protist).

EXAMPLE 3: PRODUCTION OF A CLONE PSJ17, DEFINING A RETROVIRUS MSRV-1, BY REACTION OF ENDOGENOUS REVERSE TRANSCRIPTASE WITH A PREPARATION OF VIRION DERIVED 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 only ftinetion in the presence of a retroviral RNA, linked to 9 a primer tRNA or hybridized to short strands of DMA which have already been reverse-transcribed in the retroviral particles (19). Thus, the production of specific retroviral sequences in a material contaminated with 5 cellular nucleic acids has been optimized by these authors by means of the specific enzymatic amplification of the viral RNA portions by viral reverse transcriptase activity. For this, the authors have determined the specific physicochemic&l conditions under which this 10 reverse-transcription enzymatic activity on RMAs contained in virions could be effective in vitro. These conditions correspond to the technical description o£ the procedures described below (endogenous RT reaction, purification, cloning and sequencing). 15 The molecular approach consisted in using a preparation concentrated, but not purified, virion obtained from the supernatants of a culture from the PLI-2 line prepared according to the following method: the culture supernatants are collected twice a week, 20 pre-centrifuged at 10,000 rpm for 30 minutes in order to remove the cell debris, and then frozen at -80°C or used as they are for the subsequent steps. The fresh or thawed supernatants are centrifuged on a cushion of 30% PBS-glycerol at 100,000 g (or 30,000 rpm in in LKB-HITACHI 25 rotor of type 45 T) for 2 h at 4°C. After removal o£ the supernatant, the sedimented pellet is taken up in a' small volume of PBS and constitutes the fraction of concentrated, but not purified, virion. This concentrated but not purified viral sample was used to carry out a 3 0 so-called endogenous reverse transcription reaction, as described below: a volume of 200 /il of virion purified according to the procedure described above and containing reverse transcriptase activity of about 1-5 million dpm is thawed at 37°C until a liquid phase appears, then 35 placed on ice. A 5-fold concentrated buffer was prepared with the following components: Tris-HCl pH 8.2, 500 stH; NaCl 75 mM; MgCl2 25 mM, DTT 75 mM and NP 40 0.10%. 100 fil of 5X buffer -t- 25 fil of dATP 100 mM solution + 25 fil of dTTP 100 mM solution + 25 fil of dGTP 100 mM 3 6 9 4 solution + 25 /tl of dCTP 100 mM solution + 100 pi of sterile distilled water + 200 fil of the virion suspension (RT activity of 5 million dpm) in PBS were mixed together and incubated at 42°C for 3 hours. After this incubation, 5 the reaction mixture is added directly to a phenol/chloroform/isoamyl alcohol buffered mixture (Sigma ref. P 3803); the aqueous phase is collected and a volume of sterile distilled water is added to the organic phase in order to re-extract the residual nucleic acid 10 material. The aqueous phases collected are pooled and the nucleic acids contained are precipitated by addition of 3M sodium acetate, pH 5.2 to 1/10 of volume + 2 volumes of ethanol + 1 fil of glycogen (Boehringer-Mannheim ref. 901 393) and the sample is placed at -20°C for 4 h or 15 +4°C overnight. 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 procedure described below: blunt-ended 20 DNAs with unpaired adenines at the ends were generated: a "filling-in" reaction was first carried out: 25 (il of the previously purified DNA solution were mixed with 2 fil of a 2.5 mM solution containing, in equimolar amount, dATP + dGTP + dTTP + dCTP / 1 pi of DNA polymerase T4 25 (Boehringer-Mannheim ref. 1004 786) / 5 /il of 10X "incubation buffer for restriction enzyme" (Boehringer-Mannheim ref. 1417 975) / 1 fil of 1% bovine serum albumin solution / 16 /il of sterile distilled water. This mixture was incubated for 20 minutes at 11°C. 50 /il of TE buffer 30 and 1 /il of glycogen (Boehringer-Mannheim ref. 901 393) were added thereto and the nucleic acids were then extracted with phenol/chloroform/isoamyl alcohol (Sigma ref. P 3803),, and precipitation with sodium acetate as described above. The DNA precipitated after 35 centrifugation is resuspended in 10 /il of 10 siM Tris buffer pH 7.5. 5 /il of this suspension were then mixed with 20 /il of Taq 5X buffer, 20 /il of 5 mM dATP, 1 /il (5U) of Taq DNA polymerase (Amplitaq™) and 54 /tl of sterile distilled water. This mixture is incubated for 3 6 9 2 h at 75°C with an oil film at the surface of the solution. The DMA in suspension in the aqueous solution/ withdrawn from below the oil film after incubation, is precipitated as described above and resuspended in 2 fil 5 of sterile distilled water. The DMA obtained was inserted into a plasmid using the TA Cloning™ kit. The 2 fil of DNA solution were mixed with 5 fil of sterile distilled water, 1 fil of 10-fold concentrated ligation buffer "10X LIGATION BUFFER", 2 /*1 of "pCR™ VECTOR" (25 ng/ml) and 10 1 fil of "TA DNA LIGASE". This mixture was incubated overnight at 12°C. The following steps were performed in accordance with the instructions for the TA Cloning® kit (British Biotechnology) . At the end of the procedure, the . white colonies of recombinant bacteria were picked but so 15 as to be cultured and to allow extraction of the incorporated plasmids according to the so-called "miniprep" procedure (16) . The plasmid preparation of each recombinant colony was cut with a suitable restriction enzyme and analysed on agarose gel. The plasmids possessing an 20 insert detected under UV light after labelling the gel with ethidium bromide were selected for sequencing of the insert, after hybridization with a primer complementary to the promoter Sp6 present on the cloning plasmid of the TA Cloning Kit®. The reaction prior to the sequencing was 25 then carried out according to the method recommended for the use of the "Prism ready reaction kit dye deoxyterminator cycle sequencing kit" (Applied Biosystems, ref. 401384) and automatic sequencing was performed with the "Automatic Sequencer, model 373 A" machine from Applied 30 Biosystems, according to the manufacturer's instructions.

Discriminating analysis on the computerized data banks of the sequences cloned from the DNA fragments present in the reaction mixture made it possible to reveal a sequence of retroviral type. The corresponding 35 clone PSJ17 was entirely sequenced and the sequence obtained, presented in Figure 8 and identified by SEQ ID N09, was analysed using the "Genevorks®" software on updated "Genebank®" data banks. Analysis of the data banks did not allow any identical sequence already ' 30 3 6 9 4 described to be found. Only partial homology could be found with certain known retroviral elements. The most interesting relative homology relates to an endogenous retrovirus referred to as ERV-9, or HSERV-9, according to references (20).

EXAMPLE 4: 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 carried out so as to check for the presence of -contaminants (reaction with water). The amplification consists of a step of RT-PCR according to the procedure described in Example 2, followed by "nested" PCR according to the PCR procedure described in document EP-A-0,569,272. 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: M0 02-A M003-BCD M001 POL-2 c > pol MSRV-1 POO4 POO5 RNA PSJ17 Their composition is: primer M001: GGTCITICCICAIGG (SEQ ID N020) primer M002-A: TTAGGGATAGCCCTCATCTCT (SEQ ID N021) primer M003-BCD: TCAGGGATAGCCCCCATCTAT (SEQ ID N022) primer P004: AACCCTTTGCCACTACATCAATTT (SEQ ID N023) primer POO5: GCGTAAGGACTCCTAGAGCTATT (SEQ ID N024) The "nested" amplification product obtained and referred to as M003-P004 is presented in Figure 7, and 0. - o yj ** w ^ 3 6 corresponds to the sequence SEQ ID N08.

EXAMPLE 5: 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 5 REVERSE TRANSCRIPTASE ACTIVITY A PCR technique derived from the technique published by Frohman (21) was used. The derived technique makes it possible, using a specific primer at the 3' end of the genome to be amplified, to elongate the sequence 10 towards the 5' region of the genome to be analysed. This technical variant is described in the documentation from the firm Clontech Laboratories Inc., (Palo-Alto California USA) provided with its product n5'-Ampli-FINDER™ RACE Kit", which was used on a fraction of 15 purified virion as described above.

The specific 3' primers used in the kit procedure for the synthesis of the cDNA and the PCR amplification are respectively complementary to the following MSRV-1 sequences: cDNA: TCATCCATGTACCGAA6G (SEQ ID N025) amplification: ATGGGGTTCCCAAGTTCCCT (SEQ1DN026) The products derived from the PCR were purified after purification on agarose gel, according to the conventional methods (16), and then resuspended in 10 ml 25 of distilled water. Since one of the properties of Taq polymerase consists in adding an adenine to 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 /il of DNA solution 3 0 were mixed with 5 /il of sterile distilled water, 1 /il of 10-fold concentrated ligation buffer "10X LIGATION BUFFER", 2 /il of "pCR™ VECTOR" (25 ng/ml) and 1 /il of "TA DNA LIGASE". This mixture was incubated overnight at 12 °C. The following steps were performed in accordance 35 with the instructions of the TA Cloning® kit (British Biotechnology) . At the end of the procedure, the white colonies of recombinant bacteria were picked out so as to be cultured and to allow the extraction of the incorporated plasmids according to the so-called 3 6 "miniprep" procedure (16). The preparation of plasmid from each recombinant colony was cut with a suitable restriction enzyme and analysed on agarose gel. The plasmids possessing an insert detected under XIV light after labelling the gel with ethidium bromide were selected in order to sequence the insert, after hybridization with a complementary primer of the promoter Sp6 present on the cloning plasmid of the "TA Cloning Kit®". The reaction prior to the sequencing was then carried out according to the method recommended for the use of the "Prism ready reaction kit dye deoxyterminator cycle sequencing kit" (Applied Biosystems, ref. 401384) and automatic sequencing was carried out with the "Automatic Sequencer, model 373 A" machine from Applied Biosystems, according to the manufacturer's instructions.

This technique was first applied 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 MS7GP isolate produced by the LM7PC line on the other hand: the culture supernatants are collected twice a week, precentrifuged at 10,000 rpm for 30 minutes in order to remove the cell debris and then frozen at -80°C or used as they are for the subsequent steps. The fresh or thawed supernatants are centrifuged on a cushion of 30% PBS-glycerol at 100,000 g (or 30,000 rpm in an LKB-HZTACHI rotor o£ type 45 T) 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 not purified, virions. The concentrated virus is then deposited on a gradient of sucrose in a 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 rotor with cups. 10 fractions are collected and 20 fil are taken from each fraction after homogenization, in order to assay the reverse transcriptase activity therein according to' the technique described by H. Perron (7) . The fractions containing the peak of RT activity "of type LM7" are then diluted in sterile PBS buffer and ultracentrifuged for 3 e 9 /r one hour at 35,000 rpm (100,000 g) in order to sediment the viral particles. The purified virion pellet thus obtained is then taken up in a small volume of a suitable buffer in order to extract RNA. The cDNA synthesis 5 reaction mentioned above is performed on this RNA extracted from purified extracellular virion. PCR amplification according to the technique mentioned cibove made it possible to obtain the clone Fl-11, whose sequence identified by SEQ ID N02 is presented in Figure 8. 10 This clone makes it possible to define, with the various clones sequenced beforehand, a region representative of the "pol" gene of the MSRV-1 retrovirus, as presented in Figure 9. This sequence, referred to as SEQ ID NOl, is reconstituted from various clones which 15 overlap at their ends, by correcting the artefacts linked to the primers and to the amplification or cloning techniques, which would artificially interrupt the reading frame of the system as a whole.

In Figure 9, the potential reading frame with its 20 translation into amino acids is presented below the nucleic acid sequence.

EXAMPLE 6: Capture, AMPLIFICATION AND CLONING OF A PORTION OF THE MSRV-2 GENOME USING A SEQUENCE ALREADY IDENTIFIED, IN A CULTURE INFECTED WITH MSRV-2 25 The supernatants of a cell culture expressing reverse transcriptase activity "of type LM7" similar to that described by H. Perron (7) were collected regularly for several weeks and stored in the deep-freezer at -80°C after addition of 10% glycerol. The supernatants were 30 then all thawed so as to concentrate the infective particles by ultracentrifugation and to purify them by centrifugation to the equilibrium point on a sucrose gradient; the reverse transcriptase activity was then measured in the various fractions collected on the 35 gradient according to the methodology described by H. Perron (14).

The various fractions representing the peak of reverse transcriptase activity were pooled so as to extract the nucleic acids therefrom according to a 3 a T £ U -J U procedure intended for the purification of RMA (15), but the nucleic acids extracted were not treated with DNase. A PCR amplification derived from the technique described by Shih (8) was carried out directly on this sample of nucleic acids untreated with DNase, according to an RNA amplification process as described in document EP-A-O,569,272, in a total volume of 100 p1, containing 200 ng of RNA, 1 pi of RNA Quard, 33 pmol of each mixture of primers (MOP) described by Shih (8) and identical to those used for the direct (DNA) PCR; 0.25 mM of each dNTP, 10 pi of 10X buffer, 2.5 p of Tag enzyme and 0.4 pi of RT enzyme (RT-AMV; 10 p) are also added to the samples. The amplification cycles are carried out as follows: denaturation of the RNA 65eC/10 minutes, synthesis of cDNA 50°C/8 minutes and the cycles are then identical to those of the PCR described by Shih (8) . Control reactions were carried out so as to check for the absence of contaminants (reaction on water) . The products were analysed on 10% acrylamide gel.

The sanqples 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 correspond to the sequence MSRV-2 A and to its equivalent MSRV-2B previously described in Examples 1 and 2.

Moreover, after removal of the artefactual sequences, it turns out that the other clones sequenced correspond to sequences of MSRV-1 type as described in Examples 1 and 2.

After verification of the sequences present in this nucleic acid material originating from these purified fractions containing infective particles, at least some of which are associated with reverse transcriptase activity, the nucleic acid material remaining was used to carry out a specific capture on nucleic acids carrying the MSRV2 sequence previously identified and described in Examples 1 and 2.

In a prior etep, the genetic material carrying 3 6 the MSRV2 sequence was amplified by a unidirectional PCR technique of 50 cycles using a single primer. This primer is coupled to a molecule of. biotin at its 3' end, allows unidirectional amplification from 3' to 5' and corres-5 ponds to the following sequence identified as SEQ XD N038: ' TAAAGATCTAGAATTCGGCTATAGGCGGCATCCGGCAACT 3' The capture was subsequently carried out in solution with magnetic beads coupled to avidin 10 (Dynabeads®) according to the instructions of the manufacturer (Dynal) and, after a series of washes at room temperature which allows the nucleic acids not coupled to a biotin to be removed, a PCR was carried out directly on these washed beads with a specific primer at the 3' end 15 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 20 SEQ ID N013: ' GCATCCGGCAACTGCACG 3' The PCR carried out at 35°C over 40 cycles with these primers made it possible to amplify the bio-tinylated genetic material specifically by the first step 25 of PCR and capture on the Dynabeads® beads. After cloning with the "TA cloning" kit of the DMA amplified by t&is second step of PCR and sequencing of the recombinant clones, according to the techniques described in Example 1, a sequence of 748 base pairs was obtained. This 30 nucleic acid sequence SEQ ID N012 is presented in Figure 10. This elongated sequence will be referred to herein-below as MSRV-2EL1.

The reverse sequence complementary to the primer SEQ ID M013 is present at the 3' end and is boxed in 35 Figure 10. Upstream of this primer is found the sequence already identified in the clones MSRV-2A and MSRV-2B.

The translation of this sequence into amino acids according to the 6 possible reading frames is presented in Figure 11. 1 R A 40 - An alignment of the sequence MSRV2-A (SEQ ZD NOlO) on the sequence MSRV-2EL1 (SEQ ZD N012) is presented in Figure 12. Zt is noted that the sequence MSRV-2A is strictly identical to the elongated sequence, 5 apart from a few differences in the region corresponding to the degenerate primers used to obtain MSRV-2A. This region is underlined in this figure; moreover, the hybridization region of the primer SEQ XD N013 (apart from the cloning tail) is boxed, that of the primer SEQ 10 ID N014 is presented in square brackets. The actual sequence of the MSRV-2 genome in this region is quite probably that of MSRV-2EL1, in which it has not been imposed by hybridized primers having low stringency, as is the case for MSRV-2A (and likewise MSRV-2B). 15 The sequence MSRV-2EL1 thus corresponds to a new sequenced region of the MSRV-2 genome. This was verified using novel PCR primers defined in MSRV-2EL1 and MSRV-2 A which allowed a specific amplification on the nucleic acids used for the cloning described in this example. 20 The result of interrogation of the Genebank® data bank, carried out in August 19S4, with the sequence MSRV-2EL1 shows no significant homology with genetic sequences known to date. However, interrogation of the possible translations into amino acids according to the 25 € potential reading frames of this sequence MSRV-2EL1 shows partial homologies with bacterial, viral or cellular sequences.

The absence of PCR amplification with specific primers on normal human DNA shows that this is not a 30 sequence of cellular origin. MSRV-2 is thus an infective agent which is exogenous to maui. However, the degenerate nature of the mixtures of primers, used according to variants of the technique described by Shih (8), which allowed the identification of the first sequence 35 elements, referred to as MSRV-2A and MSRV-2B, may have allowed the unforeseen amplification of a genome belonging neither to a retrovirus nor even to a gene coding for an RNA-dependent DNA polymerase. 3 6 KXAKPLE 7: CULTURING OF CELLS FROM ARTICULAR FLUIDS OF PATIENTS SUFFERING FROM RHEUMATOID ARTHRITIS (RA) AND DETECTION OF TRANSCRIPTASE ACTIVITY SIMILAR TO THAT WHICH THE CULTURE LM7 PRODUCES.

Articular fluid (AF) was withdrawn under sterile conditions from an inflamed knee joint of a patient suffering from rheumatoid arthritis. This fluid was centrifuged at +4°C at 1800 rpm for 10 minutes. After removing the supernatant and dividing it into aliquots at 10 -80°C, the cell pellet was taken up in RPMI culture medium whose composition is as follows: RPMI 1640 medium supplemented with penicillin (200,000 U/L), streptomycin (200 mg/L), L-glutamine (6 mM/1), pyruvate (1%), nonessential amino acids (1%), optionally an anti-hiunan-15 beta-interferon polyclonal antibody (10 U/ml), and 20% foetal calf serum decomplemented by incubation at 56°C for 30 minutes. These cells in suspension were transferred into a small culture flask (25 cm3) and the volume made up to 5 ml with the above-described medium in which 20 the cellr: were maintained in culture in vitro in an oven at 5% C02 and at 37°C.

After 4 days, the culture medium was replaced with fresh medium of the same composition and the medium was withdrawn and centrifuged at 1800 rpm for 10 minutes. 25 The supernatant is divided into aliquots and stored at -80°C; the pellet, consisting of cells which had not adhered to the culture flask, is taken up in culture medium and traasferred into another flask. These cells will be maintained in culture as a cell suspension. 30 The culture media are always changed at least twice a week.

After culturing for three weeks, the flask in which the first passage of cells from the pathological articular fluid was carried out contains adherent cells 35 of macrophage type and others, of fibroblast type, which exhibit a proliferation by "ranges" of mitoseB. These proliferation ranges gradually covered the lower surface of the culture flask and, at the confluent stage, these fibroblast cells were detached by scraping and divided 1 yj W v; into two 25 cm3 flasks with 5 ml of culture medium. These adherent cells were then passed and divided as many times as the potential for proliferation allowed. In effect, after the sixth passage, i.e. after culturing for about 5 two months, the fibroblast cells ceased to proliferate and the flasks were stored until the cells had degenerated.

In the flasks which were used to pass the cells in suspension, mainly cells of lymphoid type, adherent 10 cells of macrophage type or of fibroblast type sometimes adhered at a late stage. The lymphoid cells in suspension degenerated without giving rise to a proliferation and were not maintained in culture after three weeks.

The culture supernatants of all of these flaBks 15 were together stored, divided into aliquots, at -80°C.

The search for a possible retroviral agent produced by the cells of this pathological articular fluid from a patient suffering from RA first consisted in looking for reverse transcriptase activity according to 20 various physicochemical conditions allowing a nonselective detection of such an enzyme coded for by a previously unknown retrovirus, as was done by H. Perron et al., for the study of reverse transcriptase activity in culture supernatants of cells from the nervous system 25 of multiple sclerosis (7). The conditions for detecting a signal specific for reverse transcriptase activity in the culture supernatants of cells from the AF of this case of rheumatoid arthritis (RA) then proved to be very similar to those which constitute the optimum for the 30 detection of the retrovirus produced by the culture LM7 originating from a case of multiple sclerosis (MS) . It was thus sought to verify whether this activity could be detected under the same conditions as for the retrovirus MSRV1/LM7 isolated in MS, after carrying out a sedlmen-35 tation to equilibrium, on a sucrose gradient, of a concentrate of particles originating from the supernatants of cultures of the AF from RA.

A series of culture supernatants from the adherent cells of the AF of this case of RA was thawed so as i\ i to have available a total volume of at least 100 ml. These supernatants were mixed, pre-centrifuged so as to remove the cell debris and ultracentrifuged so as to sediment possible retroviral particles, and the pellet 5 thus sedimented was placed on a sucrose gradient which was centrifuged at 100,000 g so as to obtain a separation of the elements present in the ultracentrifugation pellet and to assay, after collecting fractions of an equal volume and of increasing sucrose density, the reverse 10 transcriptase activity in each of these fractions. All of these operations were carried out under the conditions described by H. Perron (14).

The result of the assay of reverse transcriptase . activity with the conditions optimized for the LM7 15 strain, in the fractions of the "RA" gradient, is presented in Figure 13. It may be seen that significant reverse transcriptase activity (greater than *rhe cut-off of 2000 dpm) is found in fractions 2 to 5, as well as in fraction 9. The activity found in the least dense frac-20 tions is poorly focused, this probably being due to an excess of material placed on the gradient (5 ml tube) . The secondary peak in the denser fraction (No. 9) quite probably consists of denser virions which have lost their envelope (capsids) as has already been observed in the 25 gradients prepared with virions produced by the LM7 line (14) .

At this stage in the work carried out on the cell cultures from AF originating from RA, it would appear that these cells produce particles possessing a reverse 30 transcriptase of the same type as that which is detected in the virions produced by the leptomeninx LM7 cells of MS.

EXAMPLE 8: CULTURE OF LYMPHOBLASTOID B CELLS FROM PATIENTS SUFFERING FROM RHEUMATOID ARTHRITIS (RA) . 35 According to a procedure which is well known to those skilled in the art, lymphoblastoid lines, immortalized by in vitro addition of the strain B95 of Epstein-Barr virus (EBV) or by the autologous strain harboured by the B lymphocytes of the patient, were 0 6 \fi vv ^ - 44 established from B lymphocytes of the peripheral blood of patients suffering from RA..

Briefly, the mononucleate blood cells were separated by centrifugation on Ficoll-Hypaque® gradient 5 (Flow/ICN), washed by centrifugation in RPMI 1640 and then taken up in a medium containing RPMI 1640 supplemented with penicillin (200,000 U/L), streptomycin (200 mg/L) , L-glut amine (2 mM/1) , pyruvate (1%), nonessential amino acids (1%), HEPES buffer (1%) and 20% 10 foetal calf serum decomplemented by incubation at 56°C for 30 minutes. These cells in suspension are transferred into a small culture flask (25 cm3) and the volume is made up to 5 xol with the above-described medium in which the cells will be maintained in culture in vitro in an 15 oven at 5% C02 and at 37°C.

The media are renewed by half twice a week: after settling the flasks placed vertically for at least 2 h, half of the supernatant medium is removed (and stored in a deep-freezer at -80°C, as soon as a line is estab-20 lished) and replaced with fresh medium in which the sedimented lymphocytes are iemulsified. When a line is established and the cell density in a flask is sufficient, the cells thus emulsified by pipette are divided between two flasks and the culture is thus split. 25 After separation on Ficoll, the lymphoid cells are placed in the presence of cyclosporin A for 10 to 20 days, so as to allow the inhibition and removal of the T lymphocytes from the culture. The reason for this is that these lymphocytes have the capacity to block the 30 immortalization of B lymphocytes present in the culture by the EBV virus.

Depending on the individual case, the culture is maintained such that after treatment with cyclosporin A, so as to wait for the possible appearance of an immortal-35 ization of B lymphocyte by the EBV strain harboured by the patient himself (if he is seropositive for this virus), or alternatively 1 ml of supernatant from the line B95 which produces EBV virions, after induction by butyric acid esters of phorbol, is added to the culture 303 0 flask so as to produce a massive infection of the B lymphocytes present by the EBV strain B95 and thus a much more probable immortalization of a few B clones.

The flasks containing the lymphocytes in suspen-5 sion are maintained in culture for at least three months, during which the appearance of immortalized B lymphocyte clones, proliferating in the form of "clusters" of cells floating in the culture medium, is monitored with a light-optical microscope.

When lympboblastoid lines are established, from patients suffering from RA, they are split regularly and maintained in continuous culture. The supernatants collected twice a week when the medium is changed are frozen at -80°C for subsequent analysis: reverse tTrans-15 criptase activity, PCR detection of the genomes of the pathogenic and/or infective agents expressed by the B cells in the culture, etc. Uses of these cultures to reveal and characterize pathogenic and/or infective agents associated with RA will be presented in the 20 following examples.

EXAMPLE 9: PRODUCTION OF CLONES MSRV-2 AND MSRV-1 BY AMPLIFICATION OF THE CONSERVED POL REGIONS OF THE RNA-DEPENDENT DNA POLYMERASE GENES ON A PREPARATION OF PATHOGENIC AND/OR INFECTIVE AGENT PURIFIED FROM A CELL CULTURE 25 OF SYNOVIAL FLUID OR OF LYMPHOBLASTOID LINE OF RA.

As in Example 1, the molecular approach consisted in using Shih's PCR technique (8) which makes it possible to amplify a relatively conserved region of the pol gene of the exogenous and endogenous retroviruses, but also of 3 0 the viruses coding tor an enzyme with reverse transcriptase (RT) activity such as, in particular, the hepatitis B virus and, implicitly, the virus of any RNA-dependent DNA polymerase or enzyme gene having sufficient sequence homologies in the regions defined by the ampli-35 fication primers used. This PCR technique was used on the nucleic acids extracted from a preparation of infective agent purified on a gradient according to the procedure described by Perron H. (14) from the supernatants of a cell culture of the pathological articular fluid (AF) 3 0 3 6 f «> 46 - from a case of rheumatoid arthritis (RA), obtained according to the procedure described in Example 7. The fractions containing the peak of RT activity measured according to the conditions optimized for the LM7 strain 5 (7) are taken up in one volume of a buffer containing guanidina thiocyanate and are stored at -80°C until the nucleic acids are extracted according to the technique described by Chomzynski P. (15).

Prior to the PCR reaction, the RNA in the sample 10 was transcribed into complementary DNA (cDNA) with so-called "random" primers (hexanucleotides as a mixture) using the "cDNA synthesis system plus" kit (Amersham), according to the manufacturer's instructions and based on an approximate value, to the nearest log factor, of the 15 amount of RNA present in the sample. Alternatively, synthesis of the cDNA may be carried out directly in the presence of the primers used for the PCR according to a one-step "RT-PCR" procedure described in document EP-A-0,569,272.

The DMA obtained after PCR amplification of the cDNA was inserted into a plasmid using the TA Cloning® kit (British Biotechnology), picked out, extracted and sequenced according to the procedures described in Examples 1 and 2.

The sequences obtained were then analysed using Mac Vector® and Geneworks® softwares on a Genebank® computerized data bank, for the nucleic acid sequences, and a Swiss Prot® computerized data bank, for the amino acid sequences deduced from the reading frames revealed 30 in the nucleic acid sequences. The analysis o£ the sequences, obtained from the sample of particles sedimen-ting at the peak of reverse transcriptase activity originating from the thawed supernatants of cell cultures of the AF from RA, revealed two types of sequence: a 35 first type of sequence, found in the minority of the clones, corresponds to sequences which are significantly homologous with the equivalent "pol" region of the MSRV-1 retrovirus previously isolated and characterized from patients suffering from MS (French patent applications - 47 36 o 92 04322, 92 13447, 92 13443, 92 01529, 94 01530, 94 01531 and 94 01532) , and a second type of sequence, found in the majority of the clones, which corresponds to sequences which are very highly homologous with the 5 sequence attributed to em infective and/or pathogenic agent, referred to as MSRV-2, previously isolated and characterized from patients suffering from MS (French patent applications 92 04322, 92 13447, 92 13443, 92 01529, 94 01530, 94 01531 and 94 01532). 10 Comparison between a sequence of type MSRV-1 amplified and cloned from viral material produced by a cell culture of AF from RA (clone "MSRVlpolPR", SEQ ID N039) and the MSRV-1 reference sequences cloned from virion produced by cells originating from patients 15 suffering from MS is presented in Figure 14.

Comparison between a sequence of MSRV-2 type amplified and cloned from infectious material produced by a cell culture of AF from RA (clone "MSRV2sPR" SEQ ID N040) and the MSRV-2 reference sequences from infectious 20 material produced by calls originating from patients suffering from MS is presented in Figure 15.

The two types of sequences found with the "reverse transcriptase" degenerate primers of Shih et al. (8) thus correspond exactly to that which has been found 25 under similar conditions, in leptomeninx cell cultures, of choroid plexus or of B lymphoblastoid lines originating from various patients suffering from MS (French patent applications 92 04322, 92 12447, 92 13443, 92 01529, 94 01530, 94 01531 and 94 01532). 30 EXAMPLE 10: VERIFICATION, USING SPECIFIC PCR PRIMERS AND A SPECIFIC HYBRIDIZATION ACCORDING TO THE ELOSA TECHNIQUE, OF THE PRESENCE OF THE SEQUENCES MSRV-1 AND MSRV2 IN THE SUPERNATANTS OF CELL CULTURES AND, DIRECTLY, IN THE BIOLOGICAL FLUIDS OF PATIENTS SUFFERING 35 FROM RA Several PCR techniques were used to detect the MSRV-1 and MSRV-2 genomes in supernatants of cultures of cells, plasmas and articular fluids of patients suffering from RA and of patients suffering from other rheumatic 0 V - u diseases (non-RA).

Extraction of the RNAs of plasma and of super-natant from cultures was carried out according to the technique described by P.Chomzynski (15), after addition 5 of a volume of buffer containing guanidinium thiocyanate to 1 ml of culture supernatant or plasma stored in a deep-freezer at -80°C after collection.

Extraction of the RNAs from articular fluid (AF) and from mononucleate blood cells (lymphocytes and monocytes) was carried out according to the following procedure: all the solutions were prepared in distilled water treated with DEPC in order to remove all trace of . RNAase; 500 /il to 1 ml of AF are thawed and 1/lOOth of the volume of a 20% SDS solution is added thereto along with proteinase K solution in a proportion of 7 fil per ml of AF; the liquid is briefly vortexed and incubated at 37 °C after addition of RNAsin (Boehringer) or RNA guard (Pharmacia); after incubation for 1 h, 1 volume of GUT buffer according to Chomzynski (15) is added and the mixture is immediately vortexed. The RNA present in the mixture is subsequently extracted according to the abovementioned procedure described by Chomzynski (15) .

For the detection of MSRV2,. the sequence MSRV- 2ELI (SEQ ID N012) made it possible to define several pairs of oligonucleotide primers which may be used for the amplification of specific DNA or RNA by the PCR technique. 3 0 The first pairs of MSRV2 primers defined below made it possible to achieve a specific detection of the MSRV-2 genome in various human cells, by a step of PCR or of RT-PCR according to an RNA amplification process as described in document EP-A-0,569,272.

The primers used are as follows: ' 5' primer, identified by SEQ ZD N014 5'GTAGTTCGATGTAGAAAGCG 3' 3' primer, identified by SEQ ZD N015 5'GCATCCGGCAACTGCACG 3' 3 6 - 49 The PCR is carried out in a succession of 35 chain-building cycles, after the step of cDNA synthesis, 1 min at 94°C, 1 min at 54°C and 1 min at 72°C.

For this example, the total RMA extracted from 5 various types of cells (15), without treatment by the DNase, was used in this RT-PCR reaction, thereby allowing a detection of RMA and DMA which are specific for the pathogen in the RMA-enriched nucleic acid extract. Indeed, our experiment shows that DMA specific for MSRV2 10 may readily be detected in the nucleic acids extracted according to the abovementioned procedure normally intended for the extraction of RMA.

The sequence of a clone "MSRV2cPR" amplified according to this technique with the PCR primers SEQ ID 15 N014 and SEQ ID M015 from cultured cells of an articular fluid from a patient suffering from RA is presented in Figure 16. Figures 17 and 18 represent alignments of the sequence of the clone "MSRV2cPR" with the MSRV2 nucleotide sequences SEQ ID NOlO and SEQ ID N012, 20 respectively, of the clones obtained from patients suffering from MS. It may be observed that the sequences originating from samples of RA or of MS are significantly homologous.

The new pairs of MSRV2 primers defined below made 25 it possible to achieve a specific detection of the MSRV-2 genome in various biological fluids from patients, by two successive steps of PCR (referred to as "nested" PCR), or of RT-PCR according to an RMA amplification process as described in document EP-A-0,569,272, it being possible 30 for the MSRV-2 sequences to be detected in the form of RMA or DMA in the biological samples.

This "nested" PCR is carried out on extracted nucleic acid samples according to that which has been described and were not treated with DNase. 35 The primers used for the first step of 40 cycles with a hybridization temperature of 48°C are as follows: 5' primer, identified by SEQ ID M044 5'GCCGATATCACCCGCCATGO 3' 3' primer, identified by SEQ ID N015 „ „ g 3 6 'GCATCCGGCAACTGCACQ 3' After this step, 10 /il of the amplification product are withdrawn and. used to perform a-. second so-called "nested" PCR amplification with primers located 5 within the region already amplified. This second step takes place over 35 to 40 cycles, with a primer hybridization temperature ("annealing") of 50°C. The reaction volume is 100 /il.

The primers used for the second step are as follows: ' primer, identified by SEQ ID N045 5'CGCGATGCTGGTTGGAGAGC 3' 3' primer, identified by SEQ ZD N046 5'TCTCCACTCCOAATATTCCQ 3' The results obtained in a series of MSRV-2 PCR carried out according to the above-described "nested PCR" technique are presented in Table ZX attached to the present description.

For MSRV-1, the amplification was carried out in 20 two steps ("nested" PCR), preceded by a step of cDNA synthesis. Furthermore, the nucleic acid sample is pretreated with DNase and a PCR control without RT (AMV reverse transcriptase) is carried out on the two amplification steps so as to verify that the RT-PCR amplifi-25 cation originates exclusively from the MSRV-1 RMA. Zn the case of control without positive RT, the starting sample of RNA divided into aliquots is again treated with DNase and re-amplified.

The procedure for treatment with DNase lacking 30 RNase activity is as follows: the extracted RNA is divided into aliquots in the presence of "RNase inhibitor" (Boehringer-Mannheim) in water treated with DEPC to a final concentration of 1 fig in 10 /il; to these 10 /il is added 1 /il of "RNase-free DNase" (Boehringesr-35 Mannheim) and 1,2 /il of buffer at pH 5 containing 0.1 M/l of sodium acetate and 51 mM/1 of MgS04; the mixture is incubated for 15 min. at 20°C and maintained at 95°C for 1.5 min. in a "thermocycler".

The first step of MSRV-1 RT-PCR may be carried 36 94 out according to a variant of the RNA amplification process as described in document EP-A-0,569,272. In particular, the step of cDNA synthesis is carried out at 42 °C for one hour, but the cDNA synthesis is better performed by following the procedure below: the RNA treated with DNase and maintained. in ice is heated at 65 °C for 10 min., then immediately plunged into ice; it is then added to a mixture maintained at 42 °C in the thermocycler intended for the PCR and containing, in IX PCR buffer, 1.5 mM MgCl2, each dNTP at 250 /*M, each primer (5' and 3' of the first step of PCR) at 300-400 nM, 10 units of RT-AMV and 2.5 units of Taq, in a final volume of 100 /il; this mixture is maintained at 42 °C for 1 to 2 h and then maintained at 95°C for 5 min, so as to denature the RT-AMV. The PCR amplification cycles then start, and continue over 40 cycles, with a hybridization phase of the primers (annealing) at 53°C for 1 min., an elongation phase at 72°C for 2 to. 3 min. and a DNA denaturation phase at 95°C for 1 min.

The primers used for this first step are as follows: ' primer 'AGGAGTAAGGAAACCCAACGGAC 3' SEQ XD N016 3' primer 'TAAGAGTTGCACAAGTGCG 3' SEQ IDN017 After this step, 10 /il of the amplification product are withdrawn and used to perform a second so-called "nested® PCR amplification with primers located within the region already amplified. The PCR amplification cycles take place over 35 to 40 cycles, with a hybridization phase of the primers ("annealing") at 53°C for 1 min., an elongation phase at 72°C for 1 to 2 min. and a DNA denaturation phase at 95°C for 1 min. The composition of the reaction mixture is the same as for the preceding step, except that the RT-AMV is not added this time.

The primers used for this second step are as follows: ' primer, identified by SEQ ID N018 - 52 3 6 ' TCAGGGATAGCCCCCATCTAT 3' 3' primer, identified by SEQ ID N019 5 ' AACCCTTTGCCACTACATCAATTT 3' The sequence of a clone "MSRVlnPR" amplified 5 according to the above-described technique with the PCR primers SEQ XD N016 and SEQ ID N017, followed by SEQ XD NOl 8 and SEQ ID N019 from RNA of an articular fluid of a patient suffering from RA is presented in Figure 19. Figure 20 represents the alignment of the sequence of the 10 clone "MSRVlnPR" with the MSRV1 nucleotide sequence SEQ ID NOOl of the reference clone obtained in patients suffering from MS. Here also, it may be observed that the sequences originating from RA or from MS are significantly homologous.

To date, the other MSRV1 and MSRV2 sequences amplified according to these PCR techniques from samples of RA were also revealed to be identical to or homologous with the sequences obtained in patients suffering from MS.

In a series of MSRV-1 PCR carried out according to the above-described nested "RT-PCR" technique, the results presented in Table I attached to the present description were obtained.

The MSRV-1 retroviral genome and the MSRV-2 25 genome are thus indeed found in the biological fluid samples of patients suffering from RA. Other results obtained on wider series confirm these results.

Moreover, the specificity of the sequences amplified by these PCR techniques may be verified and 30 evaluated by the "ELOSA" technique as described by F. Mallet (22) and in document FR-2,663,040.

For MSRV-1, the products of the above-described nested PCR may be tested in two ELOSA systems, which makes it possible to detect separately a consensus A and 35 a consensus B + C + D of MSRV-1, corresponding to the sub-f ami lies described in Example 2 and Figures 2, 3 and 4. Indeed, sequences similar to the consensus B + C + D are found essentially in the samples of RNA originating from MSRV-1 virions purified from cultures or amplified 3036 94 in extracellular biological liquids from RA or MS patients, whereas the sequences similar to the consensus A are essentially found in normal human cellular DMA.

The ELOSA/MSRV-1 system for the capture and 5 specific hybridization of the PCR products of the subfamily A uses a capture oligonucleotide cpVIA with a 5' amine bond according to a technique described in the document FR-A-2,663,040, and a detection oligonucleotide dpVIA coupled to peroxidase according to a technique 10 described in document FR-A-2,663,040 (or optionally coupled to biotin) respectively having the sequence: cpVIA 5' GATCTAQOCCACTTCTCAGGTCCAGS 3' SEQ ID N047 dpVIA 5' CATCTITTTGGICAGGCAITAGC 3' SEQ ID N048 The ELOSA/MSRV-1 system for the capture and 15 specific hybridization of the PCR products of the subfamily B+C+D uses the same detection oligonucleotide dpVIA coupled to peroxidase according to the technique described above (or optionally coupled to biotin) and a capture oligonucleotide cpVlB with a 5' amine bond accor-20 ding to the technique described above, having the sequence: cpVIB 5' CTTGAGCCAGTTCTCATACCTGGA 3' SEQ ID N049 As can be seen in Table I, such a technique was applied to the amplified PCR products from biological 25 fluids of patients suffering from various rheumatological diseases: all the patients suffering from RA, who had an amplified DNA band of the expected size detectable on agarose gel labelled with BET, were positive for B + C + D type MSRV1 and negative for MSRV1-A; only a single non-30 RA control had an amplified band visible on gel labelled with BET, of an apparently correct size; the corresponding PCR product proved to be negative on ELOSA MSRV1-A and MSRV1-BCD, along with all of the PCR products of the other non-RA controls; after cloning and sequencing of 35 this amplified product, it turned out that it corresponded to an artefactual sequence bearing no relationship with MSRV1.

The ELOSA MSRV1 technique coupled with the nested RT-PCR defined above thus makes it possible to perform a T f* X <? 0 5 v very sensitive and very specific detection test for a retrovirus MSRV 1 in patients' biological fluids.

It may thus also be envisaged, by means of the discoveries made and the methods developed by the inventors within the context of rheumatoid arthritis, to carry out a diagnosis of MSRV-1 and/or MSRV-2 infection and/or reactivation and to evaluate a therapy in RA based on its effectiveness to "negate" the detection of these agents in the biological fluids from the patients. Furthermore, the early detection in individuals not yet exhibiting rheumatological signs of RA could make it possible to install a treatment which is proportionately more effective on the subsequent clinical evolution the more it precedes the lesional stage which corresponds to the appearance of the articular attack. Now, a diagnosis of RA cannot to date be established before the installation of an inflammatory or even lesional symptomatology, and thus no treatment is installed before the emergence of an already notable clinical indicator of an articular attack.

The diagnosis of an MSRV-1 and/or MSRV-2 infection emd/or reactivation in man is thus a determining factor, and the present invention provides the means for such a diagnosis both within the context of a neurological (MS) or rheuma tological (RA) symptomatology which are associated, or alternatively within the context of an infection/reactivation which is pre-symptomatic or not yet associated with a well-characterized clinic.

Besides carrying out a diagnosis of MSRV-1 and/or MSRV-2 infection and/or reactivation, it is thus possible to evaluate a therapy in MS, RA or any other associated clinical symptomatology on the basis of its effectiveness to "negate" the detection of these agents in the biological fluids of the patients. - 55 3 6 9 TABLE I Results of the detection, by PCR followed by a hybridization according to. the so-called ELOSA technique, of the MSRV-1 genomes in the biological fluids of patients suffering from RA and other rheumatological diseases.

Diagnosis Number tested Saaqple MSKV-1 MSKV-1 MSKV-1 nastad SLOSH KMSli RT-PCR sub-type sub-typ* (BET A B band) (B+C+D) Rheumatological non-RA control jut cell culture medium - 0 + - 0 + - 0 + RA AF call culture niariiwn -5 + -0 + -5 + Rheumatological non-RA control articular fluid 8 - 0 + 8 -0 + 8 -0 + RA articular fluid 3 -2 + -0 + 3 -2 + Rheumatological non-RA control Plana 6 -1 + 7 -0 + 7 -0 + RA Plasma 4 - 6 - 4 - Rheumatological non-RA control mononucleate! blood calls (dry pellet) 2 + 4 - 0 + 0 + 4 - 0 + 2 + 4 - 0 + RA mononucleate 0 - 2 - blood cells (dry pellet) 2 + 0 + 0 -2 + T & r- i ; - 56 -TABLE II Results of the detection., by PCR followed by a hybridization according to the so-called ELOSA technique, of the MSRV-2 genomes in the biological fluids of patients suffering from RA and other rheumatological diseases.

Diagnosis Number tested Saaple Rheumatological 4 non-RA control RA Rheumatological 5 non-RA control Articular fluid Articular fluid Plasma MSRV-2 nested RT-PCR (BET band) 4 - 0 + 1 - 4 + 4 - 1 + positive patient polytransfused patient RA Plasma 6 - 2 + m - 57 -SEQUENCE LISTING 3 6 ? (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: BIOMERIEUX (B) STREET: NONE (C) CITY: MARCY L'ETOILE (E) COUNTRY: FRANCE (F) POSTAL CODE: 69280 (ii) TITLE OF THE INVENTION: MS EXTENSION 10 (ill) NUMBER OF SEQUENCES: 49 (iv) COMPUTER-READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 1158 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDMA (xi) SEQUENCE DESCRIPTION: SEQ ID NO,'; 1: CCCTTTGCCA CTACATCAAT TTTAGGAGTA AGGAAA'JCCA ACGJACAGTG GAGGTTAGTG 60 CAAGAACTCA GGATTATCAA TGAGGCTGTT GTTCCTCTAT ACCCAGCTGT ACCTAACCCT 120 TATACAOTQC TTTCCCAAAT ACCAGAGGAA GCAGAGTGQT TTACAGTCCT GGACCTTAAG 180 QATQCCTTTT TCTGCATCCC TGTAC8TCCT GACTCTCAAT TCTTGTTTGC CTTTQAAGAT 240 CCTTTGAACC CAACGTCTCA ACTCACCTGG ACTGTTTTAC CCCAAGGGTT CAGGGATAGC 300 CCCCATCTAT TTCCCCAOOC ATTAGCCCAA GACTTOAGTC AATTCTCATA CCTOGACACT 350 CTTGTCCTTC AGTACATGGA TOATTTACTT TTAOTCGCCC GTTCAGAAAC CTTQTQCCAT 420 CAAQCCACCC AAGAACTCTT AACTTTCCTC ACTACCTGTG OCTACAAGOT TTCCAAACCA 480 AAGGCTCGGC TCTQCTCACA GGAGATTAGA TACTHAGGGC TAAAATTATC CAAAGGCACC 540 AQGGCCCTCA OTOAGOAACa TATCCAGCCT ATACTGGCTT ATCCTCATCC CAAAACCCTA £00 AAGCAACTAA OASQGTTCCT TGGCATAACA OOTTTCTQCC GAAAACAGAT TCCCAGGTAC 650 ASCCCAATAS CCAGACCATT ATATACACTA ATTANGOAAA CTCAOAAAGC CAATACCTAT 720 TTAGTAAOAT OOACACCTAC AGAAGTGGCT TTCCAOOCCC TAAAGAAGQC CCTAACCCAA 780 GCCCCAQTQT TCAGCTTGCC AACAGOQCAA QATTTTTCTT TATATGCCAC AGAAAAAACA 840 OGAATAOCTC TAGGAOTCCT TACOCAOOTC TCASGGATGA GCTTOCAACC CGTOOTATAC 900 CTGAGTAAGQ AAATTOATGT AGTGGCAAAG GGTTGGCCTC ATNGTTTATG GGTAATGGNG 960 OCAOTAOCAO TCTNAQTATC TGAAGCAGTT AAAATAATAC AGG3AAGAGA TCTTNCTGTG1020 TSGACATCTC ATSATGTOAA CGGCATACTC ACTGCTAAAG GAGACTTGTO GTTGTCAGAC1080 AACCATTTAC TTAANTATCA QOCTCTATTA CTTOAAOAGC CAGTGCTGNG ACTGCGCACT114 0 TGTOCAACTC TTAAACCC 1158 3 6 (2) INFORMATION FOR SEQ ID NO: 2: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 297 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CCCTTTGCCA CTACATCAAT TTTAQQAGTA AGGAAACCCA ACGGACAGTG GAGGTTAGTG 60 10 CAAGAACTCA GQATTATCAA TGAGQCTGTT GTTCCTCTAT ACCCAQCTGT ACCTAACCCT 120 TATACAGTOC TTTCCCAAAX ACCAGAGGAA GCAGAGTGGT TTACAGTCCT GOACCTTAAO 180 GATGCCTTTT TCTGCATCCC TGTACGTCCT GACTCTCAAT TCTTOTTTGC CTTTOAAGAT 240 CCTTTOAACC CAACGTCTCA ACTCACCTOG ACTGTTTTAC CCCAAOQGTT CAAGQOA 257 (2) INFORMATION FOR SEQ ID HO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 85 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GTTTAQGSAT ANCCCTCATC TCTTTQQTCA GGTACTOGCC CAAOATCTAG GCCACTTCTC 60 AGGTCCAQSN ACTCTGTYCC TTCAQ 85 (2) INFORMATION FOR SEQ XD NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 86 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 303 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GTTCAGGGAT AGCCCCCATC TATTTGGCCA GGCACTAOCT CAATACTTQA GCCAGTTCTC ATACCTGGAC AYTCTYGTCC TTCQGT 85 (2) INFORMATION FOR SEQ ID NO: 5s (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 85 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GTTCARRGA TAGCCCCCATC TATTTGGCCW R07ATTAGCC CAAQACTTGA GYCAATTCTC SO ATACCTGGA CACTCTTGTCC TTXRO 85 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 85 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO; 6: GTTCAGGOAT AGCTCCCATC TATTTGGCCT OQCATXAACC CGAOACTTAA GCCAQTTCTY 50 ATACGTGGAC ACTCTTGTCC TTTG3 85 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 111 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear •61 - 3 0 3 (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GTGTTGCCAC AGQOGTTTAR ROATANCYCY CATCTMTTTG GYCWRGYAYl" RRCYCRAXAY 60 YTRRGYCAVT TCTYAKRYSY RGSNAYTCTB KYCCTTYRGT ACATGGATGA C 111 (2) INFORMATION FOR SEQ ID NO: 8: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH; 645 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single 10 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID MO: 8: TCAGGGATAG CCCCCATCTA TTTGGCCAGG CATTAGCCCA AGACTTGAGT CAATTCTCAT 60 ACCTQQACAC TCTTGTCCTT CAGTACATGG ATGATTTACT TTTAGTCSCC CGTTCAGAAA 120 CCTTGTGCCA TCAAGCCACC CAAGAACTCT TAACTTTCCT CACTACCTGT GGCTACAAGG 180 TTTCCAAACC AAAGGCTCGG CTCTGCTCAC AQOAGATTAG ATACTNAGGG CTAAAATTAT 240 CCAAAGGCAC CAGGGCCCTC AGTGAGGAAC QTATCCASCC TATACTGGCT TATCCTCATC 300 CCAAAACCCT AAAGCAACTA ASAGGGTTCC TTGGCATAAC AGOTTTCTGC CGAAAACAOA 360 TTCCCAGGTA CASCCCAATA GCCAGACCAT TATATACACT AATTANOQAA ACTCAGAAAO 420 CCAATACCTA TTTAGTAAGA TOGACACCTA CAGAAGTGGC TTTCCAGGCC CTAAAGAAGQ 480 CCCTAACCCA AGCCCCAGTQ TTCAGCTTQC CAACAGGGCA AGATTTTTCT TTATATGCCA 540 CAGAAAAAAC AGGAATAGCT CTAGGAGTCC TTACGCAGGT CTCAGGGATG AGCTTGCAAC 600 CCGTGGTATA CCTQAGTAAQ GAAATTGATG TAGTGGCAAA GGGTT 645 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 741 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear 3 0 (ii) MOLECULE TYPE: cDNA 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CAAQCCACCC AAGAACTCTT AAATTTCCTC ACTACCTGTG GCTACAAGGT TTCCAAACCA AAGQCTCAGC TCTGCTCACA GGAGATTAGA TACTTAGGGT TAAAATTATC CAAAQGCACC 60 120 - 62 3 6 9 AGGGGCCTCA GTGAGQAACO TATCCAGCCT ATACTGGGTT ATCCTCATCC CAAAACCCTA 180 AAGCAACTAA GAGGGTTCCT TAGCATGATC AGGTTTCTGC CGAAAACAAG ATTCCCAGOT 240 ACAACCAAAA TAGCCAGACC ATTATATACA CTAATTAAGG AAACTCAGAA AQCCAATACC 300 TATTTAQTAA GATGGACACC TAAACAGAAG GCTTTCCAGG CCCTAAAGAA GOCCCTAACC 360 CAAGCCCCAG TGTTCAGCTT GCCAACAGGG CAAGATTTTT CTTTATATGG CACAGAAAAA 420 ACAGGAATCG CTCTAGGAGT CCTTACACAG GTCCGAGGGA TGAGCTTGCA ACCCGTQGCA 4B0 TACCTGAATA AGGAAATTGA TGTAGTGGCA AAGGOTTGGC CTCATNGTTT ATGGGTAATG 540 GNGGCAGTAG CAGTCTNAGT ATCTGAAGCA GTTAAAATAA TACAGGGAAG AGATCTTNCT 600 GTGTGGACAT CTCATGATGT GAACGGCATA CTCACTGCTA AAGGAGACTT GT3QTTGTCA 660 GACAACCATT TACTTAAHTA TCAGGCTCTA TTACTTGAAG AGCCAGTGCT GNGACTGCGC 720 ACTTGTGCAA CTCTTAAACC C 741 (2) INFORMATION FOR SEQ XD NO: 10: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 93 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: TSGAAAQTGT TGCCACAQGG CGCTGAAGCC TATCGCGTGC AGTTGCCQGA TGCCGCCTAT 60 AGCCTCTACA TGGATGACAT CCTGCTGGCC TCC 93 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 96 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: TTGGATCCAG TGYTGCCACA GGSCGCTQAA GCCTATCGCG TGCAQTTGCC GGATGCCGCC TATAGCCTCT ACGTGGATGA CCTSCTGAAQ CTTGAG 60 96 (2) INFORMATION FOR SEQ ID MO: 12: 303 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 748 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: TQCAASCTTC ACCQCTTOCT G0ATGTAG3C CTCAOTACCG GNGTGCCCCG CGCGCTQTAG 60 TTCGATGTAG AAAQCGCCCO GAAACACGCG GGACCAATQC GTCOCCAGCT TGCGCGCCAG 120 CGCCTCGTTG CCATTGOCCA GCOCCACGCC GATAXCACCC QCCATOOCGC CGGAGAGCGC 180 CAGCAGACCG GCGGCCAGCG GCGCATTCTC AACQCCSGQC TCGTCGAACC ATTCG3GQOC 240 GATTTCCGCA CGACCGCGAT GCTOGTTGOA GAOCCAGGCC CTGGCCAGCA ACTGGCACAG 300 GTTCAGGTAA CCCTGCTTQT CCCGCACCAA CAOCAGCAGG CGGOTCGGCT TGTCGCGCTC 360 GTCQTGATTG GTGATCCACA CGTCAGCCCC GACGATOGGC TTCACGCCCT TGCCACGCGC 420 TTCCTTGTAG ANGCGCACCA GCCCGAAGOC ATTQOCGAGA TCOGTCAQCG CCAAGGCGCC 480 CATGCCATCT TTGGCGGCAG CCTTGACGOC ATCGTCGAGA CGGACATTGC CATCGACOAC 540 GGAATATTCG GAGTGGAGAC GGAGGTOGAC GAAGCGCGGC GAATTCATCC GCQTATTQTA 600 ACGGGTGACA CCTTCCOCAA AQCATTCCOO AC8T0CCC0A TTGACCCGGA GCAACCCCOC 660 ACGGCTGCOC aOGCAOTTAT AATTTCOGCT TACGAATCAA COGSTTACCC CAOGOCOCTG 720 AAGCCTATCO COTOCAOTTG CCOOATOC 74B (2) INFORMATxCTT FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GCATCCGGCA ACTGCACG 18 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: 3 6 9 (A) LENGTH: 20 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GTAGTTCGAT GTAGAAAGCG 20 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GCATCCGGCA ACTGCACG 18 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: AGGAGTAAGG AAACCCAACG GAC 23 (2) INFORMATION FOR SEQ ZD NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: TAAGAGTTGC ACAAGTGCG 19 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single 15 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: TCAGGGATAG CCCCCATCTA T 21 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear I (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19 66 - 3 $ aaccctttgc cactacatca attt 24 (2) INFORMATION FOR SEQ ZD NO: 20: (£) SEQUENCE CHARACTERISTICS« (A) LENGTH: 15 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS t single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) CHARACTERISTICS: (B) LOCATION: 5, 7, 10, 13 (D) OTHER INFORMATION: G represents inosine (i) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: GGTCGTGCCG CAGGG 15 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: TTAGGGATAG CCCTCATCTC T 21 (2) INFORMATION FOR SEQ ZD NO: 22: (i) (a) SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs 3 6 9 (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: TCAGGGATAG CCCCCATCTA T 21 (2) INFORMATION FOR SEQ ID HO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: 15 AACCCTTTGC CACTACATCA ATTT 24 (2) INFORMATION FOR SEQ ID NO: 24; (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleotide 2 0 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: GCGTAAGGAC TCCTAGAGCT ATT l 23 I -es- ts) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: TCATCCATGT ACCGAAGG 18 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single 15 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: ATGGGGTTCC CAAGTTCCCT 20 (2) INFORMATION FOR SEQ ID NO: 27; (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: GCCGATATCA CCCGCCATGG 20 (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: 10 GCATCCGGCA ACTGCACG 18 (2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: CGCGATGCTG GTTGGAGAGC 20 (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single 25 (D) TOPOLOGY: linear # '30 3 6 9 ~ - 70 - (ii) MOLECULE TYPE: CDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: TCTCCACTCC GAATATTCCG 20 (2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: GATCTAGGCC ACTTCTCAGG TCCAGS 26 (2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) CHARACTERISTICS: (B) LOCATION: 6/12, 19 (D) OTHER INFORMATION: G represents inosine (i) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: CATCTGTTTG GGCAGGCAGT AGC 23 3 (2) INFORMATION FOR SEQ ID MO: 33: (?) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: CTTGAGCCAG TTCTCATACC TGGA 24 (2) INFORMATION FOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: AGTGYTRCCM CARGGCGCTG AA 22 (2) INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: 3 5 GMGGCCAGCA GSAKGTCATC CA 22 (2) INFORMATION FOR SEQ ID NO: 36: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: 10 GGATGCCGCC TATAGCCTCT AC 22 (2) INFORMATION FOR SEQ ID NO: 37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: AAGCCTATCG CGTGCAGTTG CC 22 (2) INFORMATION FOR SEQ ID NO: 38; (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single 25 (D) TOPOLOGY: linear 3 0 5 0 9 k - *73 - (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: TAAAGATCTA 6AATTC66CT ATAGGCGGCA TCC6GCAA6T 40 (2) INFORMATION FOR SEQ ID NO: 39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 126 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: GTOCTACCAC AQGGQTTCAO GOATAOCTCC CATCTATTTO OCCTOXCATT AACCCQAQAC SO TTAAQCCAOT TCTCATACQT GOACXCTCTT OTCCTTTOOT ACOTOQATOA CATCCTQCTO 120 OCCTCC 126 (2) INFORMATION FOR SEQ ID NO: 40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 87 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single 20 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: QTQCTGCCCC AOOOCaCTOA AQCCTATCOC QTOCAOTTQC COOATOCCQC CTATAQCCTC 60 TACQTOQATQ ACCTQCTSCT QGCCTCC 87 (2) INFORMATION FOR SEQ ID NO: 41: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH* 705 base pairs # 3 6 h. - 74 (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION : SEQ ID NO: 41: gtagttcgat gtagaaagcg cccggaaaca cgcqggacca atscgtcgcc agcttgcqcg 60 ccagcgcctc gttgccattg 8ccagcgcca cgccgatatc acccgccatg gccgccggaq 120 agcgccaqca gaccggcggc caqcggcgca ttctccaacg ccgggctcgt cgaatcattc 180 gggggcgatt tccccacgac cgcgatgctg gttggagagc caggccctqg ccagcaactg 240 qcacaggttc aggtaacccc tgcttgtccc cocacccaac ascagcagqc qqgtcgqctt 300 gtcgcqctcq tccgtgattg gtggatccac aacgtcascc ccoacgatog gcttcacocc 360 cttgccacgc gcttccttgt aqaagcgcac cagcccgqaa ggcattggco agatcgqtca 420 agcgccaaqq nsccccatgc catctttggc ggcaqgcctt gacqgcatcg tcgagacgga 480 cattgccatc gaccgacgga atattcqgag tggagacgqa gqtggacgaa qcqcggcgaa 540 ttcatccgcq tattgtaacg ggtgacacct tccccaaaqc attccgggcg tgcccgattq 600 acccggagca accccqcacg gctgcqcggg cagttataat ttcsgcttac gaatcaacgg 660 gttaccccaq ggcgctgaag cctatcgcgt gcagttgcca gatgc 705 (2) INFORMATION FOR SEQ ID NO: 42: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 648 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: cagggtatao cccccatcta tttggccagg cattaqccca aoacttoaqc cagttctcat 60 acctggacac tcttgtcctt cagtatatgg atgatttact tttagtgacc cattcagaaa 120 ccttgtqatg tcaagccaca caagtgctct taactttcct ctttacctct ggctacaagg 180 ttttcaaacc aaaqgctcag ctctgctcac agcaggttaa atatttaggg ctaaaattat 240 30 ccaaaggcac caqgqccctc agtoaggaac gtatccaocc tatactggct tatcttcatc 300 ccaaaaccct aaagcaacta agasgqttcc ttggcataac aogcttctqc tqaatatqqa 360 ttcccaggtt yggtgaaata gccaggccat taaatacact aattaaggaa actcagaaag 420 ccaataccca tttagtaaga tggacatctg aagcacaatc aqctttccag gcactaaaga 480 aagccctaac ccaagcccca gtgttaagct tqccaacagg gcaaoacttt tctttatatg 540 35 tcacagaaaa aataggaata gctctaqgag tccttacaca gotctqaggg acaagcttgc 600 aacccqtggc atatctgagt aaggarrctg atgtagtggc aaagqgtt 648 * (2) INFORMATION FOR SEQ ID NO: 43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: CAGGGTATAO CCCCCATCTA TTTOOCCASO CATTAOCCCA AOACTTGAOC CAQTTCTCAT 60 10 ACCTGQACAC TCTTQTCCTT CAOTATATGO ATGATTTACT TTTAGTGACC CATTCAGAAA 120 ccttgtqatg TCAAGCCACA CAAGTGCTCT TAACTTTCCT CTTTACCTCT GGCTACAAGG 180 TTTTCAAACC AAAQGCTCAG CTCTGCTCAC AGCAOGTTAA ATATTTAGGG CTAAAATTAT 240 CCAAAGGCAC CAGSSCCCTC AGTGAGGAAC GTATCCAQCC TATACTGGCT TATCTTCATC 300 CCAAAACCCT AAAGCAACTA AGAOOOTTCC TTGGCATAAC AGGCTTCTGC TGAATATGGA 360 15 TTCCCAGGTT YGGTGAAATA GCCAGOCCAT TAAATACACT AATTAAGGAA ACTCAGAAAG 420 CCAATACCCA TTTAGTAAGA TGGACATCTG AAGCACAATC AGCTTTCCAG GCACTAAAGA 480 AAGCCCTAAC CCAAGCCCCA GTGTTAAGCT TQCCAACAGG GCAAGACTTT TCTTTATATG 540 TCACAGAAAA AATAQGAATA GCTCTAGGAQ TCCTTACACA QGTCTSAQGQ ACAAGCTTGC 600 AACCCGTGQC ATATCTGAGT AAGOABRCTG ATGTAOTQGC AAAGOSTT 648 (2) INFORMATION FOR SEQ ID NO: 44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single 25 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: GCCGATATCA CCCGCCATGG 20 (2) INFORMATION FOR SEQ ID NO: 45: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45: CGCGATGCTG GTTGGAGAGC 20 (2) INFORMATION FOR SEQ ID NO: 46: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46: 15 TCTCCACTCC GAATATTCCG 20 (2) INFORMATION FOR SEQ 3D NOs 47: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: GATCTAGGCC ACTTCTCAGG TCCAGS 26 # 5 0 y4 (2) INFORMATION FOR SEQ ZD 210: 48: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48: CATCTITTTG GICAQQCAIT AGC 23 (2) INFORMATION FOR SEQ XD NO: 49: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single 15 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: CTTGAGCCAG TTCTCATACC TGGA 24 # ^ £ 0 /.

I.J \J O' *T - 78 -BIBLIOGRAPHY (1) Sauvezio B. et al., in "Polyarthrite rhumatoide, aspects actuals et perspectives [Rheumatoid arthritis, current aspects and perspectives]", Sany J., 1-13, Collection Mfidecine-Sciences Flammarion, Paris, 1987 (2) Kahen A., Virus et polyarthrite rhumatoide [Virus and rheumatoid arthritis] , in "Polyarthrite rhumatoide, aspects actuels et perspectives [Rheumatoid arthritis, current aspects and perspectives] " , Sany J., 1-13, Collection MSdecine-Sciences Flammarion, Paris, 1987 (3) Fujinami R.S. and Oldstone M.B.A., ed. Current Topics in Microbiology and Immunology, 145. Berlin, Springer Verlag, 1989 (4) Acha-Orbea H. and Palmer E., Mis -a retrovirus exploits the immune system- Immunology Today 1991; 12, 271-276 (5) Cole B.C. and Atkin C.L., The irycoplasma arthritidis 20 T-cell mitogen, MAM : a model superantigen.

Immunology Today 1991 ; 12, 271-276 (6) Posnet D.N., Do superantigens play a role in autoimmunity ? Semin. Immunol. 1993; 5, 65-72 (7) Perron H. et al., Res. Virol. 1989; 140, 551-561 25 (8) Shih A., Misra R. and Rush M.G., Res. Virol. 1989; 63, 64-75 (9) Fields and Knipe, Fundamental Virology 1986, Rev Press N.Y. (10) Nielsen P.E. et al. Science 1991; 254, 1497-1500 (11) Mani atis et al.. Molecular Cloning, Cold Spring Harbor, 1982 (12) Southern. E.M., J. Mol. Biol. 1975; 98, 503 (13) Dunn A.R. and Hassel J.A., Cell 1977; 12, 23 (14) Perron H. et al.. Res. Vir. 1992; 143.' 337-350 (15) Chamzynski P. and Sacchi N., Analytical Biochemistry 1987; 162, 156-159 (16) Sambrook J., Fritsch E.F., and Maniatis T., Molecular cloning, a laboratory manual, Cold Spring 3 a 7. ft n /. U O* U v3? 4 Harbor, Laboratory Press, 1989 (17) Meyerhaxxs et al., Cell 1989; 38, 901-910 (18) Linial H.L. and Miller A.D., "Current topics in microbiology and isnounobiology. Retroviruses, strategies o£ replication" vol. 157. 125-152; Swanstrom R. and Vogt P.K., editors. Springer-Verlag, Heidelberg 1990 (19) Lori P. et al., J. Virol. 1992; 6£, 5067-5074 (20) La Mantia et al.. Nucleic Acids Research 1991; 19, 10 1513-1520 (21) Frohman et al., Proc. Natl. Acad. Sci. USA 1988; 85, 8998-9002 (22) Mallet F. et al.. Journal of Clinical Microbiology • 1993; 31, 1444-1449 3b y WO 96/28552 - 80 - PCT/FR96/00360

Claims (37)

1. Use of a viral material, in the purified or isolated state, possessing reverse transcriptase activity, related to a family of endogenous retroviral 5 elements, as derived from a viral strain possessing reverse transcriptase activity, chosen from the strains referred to respectively as POL-2, filed on 22.07.1992 with the ECACC under the access number V92072202, and MS7PG filed on 08.01.93 with the ECACC under the access 10 number V93010816, and from the variant strains thereof consisting of viruses comprising at least one antigen which is recogniz'ed by at least one antibody directed against at least one antigen corresponding to either of the viruses of the abovementioned viral strains POL-2 and 15 MS7PG, in order to obtain a diagnostic, prophylactic or therapeutic composition for detecting, preventing or treating an infection by the said viral material associated with rheumatoid arthritis, or a reactivation of the said material. 20

2. Use of a viral material, in the purified or isolated state, possessing reverse transcriptase activity, related to a family of endogenous retroviral elements, as produced by a cell line chosen from the lines referred to respectively as PLI-2 filed on 25 22.07.1992 with the ECACC under the access number 92072201, and LM7PC filed on 08.01.93 with the ECACC \inder the access number 93010817, or by any infected cell culture capable of producing a virus comprising at least one antigen which is recognized by at least one antibody 30 directed against at least one antigen corresponding to either of the viruses produced by the abovementioned lines PLI-2 and LM7PC, in order to obtain a diagnostic, prophylactic or therapeutic composition for detecting, preventing or treating an infection by the said viral 35 material associated with rheumatoid arthritis, or a reactivation of the said material.

3. Use of a viral material whose genome comprises a nucleotide sequence chosen from SEQ ID NOl, SEQ ID N02, SEQ ID N03, SEQ ID N04, SEQ ID N05, SEQ ID N06, SEQ ID - 81 30 3 6 9 4 N07, SEQ ID N08, SEQ XD N09, SEQ ID N039, SEQ ID N042 and SEQ ID N043, complementary sequences thereof and equivalent sequences thereof, in particular the nucleotide sequences having, for any sequence of 100 contiguous 5 monomers, at least 50% and preferably at least 70% homology with a nucleotide sequence chosen from SEQ XD NOl, SEQ ID N02, SEQ ID N03, SEQ ID N04, SEQ ID NOS, SEQ ID N06, SEQ ID N07, SEQ ID N08, SEQ ID N09, SEQ ID N039, SEQ ID N042 and SEQ ID N043 and complemen-10 tary sequences thereof, in order to obtain a diagnostic, prophylactic or therapeutic composition for detecting, preventing or treating an infection by the said viral material associated with rheumatoid arthritis, or a reactivation of the said material. 15

4. Use of a retroviral material, the pol gene of whose genome comprises an equivalent nucleotide sequence, and especially having at least 50% homology, preferably at least 65% homology, with a nucleotide sequence belonging to the pol gene of the genome of the retrovirus ERV-9 2 0 or HSERV-9, in order to obtain a diagnostic, prophylactic or therapeutic composition for detecting, preventing or treating an infection by the said viral material associated with rheumatoid arthritis, or a reactivation of the said material. 25

5. Use of a retroviral material, the pol gene of whose genome codes for a peptide sequence having at least 50% and preferably at least 70% homology, with a peptide sequence coded for by the pol gene of the genome of the retrovirus ERV-9 or HSERV-9, in order to obtain a diag-30 nostic, prophylactic or therapeutic composition for detecting, preventing or treating an infection by the said viral material associated with rheumatoid arthritis, or a reactivation of the said material.

6. Use of a retroviral material, the pol gene of 35 whose genome codes ^for a peptide sequence having, for any contiguous sequence of at least 30 amino acids, at least 50% and preferably at least 70% homology with a peptide sequence coded for by a nucleotide sequence chosen from SEQ ID NOl, SEQ ID N02, SEQ ID N03, SEQ ID NQ4, SEQ ID » 50 36 9 N05, SEQ IS NO6, SEQ ID N07, SEQ ID N08, SEQ ID N09, SEQ ID N039, SEQ ID N042 and SEQ ID N043 and complementary sequences thereof, In order to obtain a diagnostic, prophylactic or therapeutic composition for detecting, 5 preventing or treating an infection by the said viral material associated with rheumatoid arthritis, or a reactivation of the said material.

7. Use of a nucleotide fragment whose nucleotide sequence comprises a nucleotide sequence chosen from 10 SEQ ID NOl, SEQ ID N02, SEQ ID N03, SEQ ID N04, SEQ ID N05, SEQ ID N06, SEQ ID N07, SEQ ID N08, SEQ ID N09, SEQ ID N039, SEQ ID N042 and SEQ ID N043, complementary sequences thereof and equivalent sequences thereof# in particular the nucleotide sequences having, for any 15 sequence of 100 contiguous monomers, at least 50% and preferably at least 70% homology with a sequence chosen from SEQ ID NOl, SEQ ID N02, SEQ ID N03, SEQ ID N04, SEQ ID N05, SEQ ID N06, SEQ ID N07, SEQ ID N08, SEQ ID N09, SEQ ID N039, SEQ ID N042 and SEQ ID N043 and cosgtle-20 mentary sequences thereof, in order to obtain a diagnostic, prophylactic or therapeutic composition for detecting, preventing or treating an infection by the said viral material associated with rheumatoid arthritis, or a reactivation of the said material. 25

8. The use of a specific primer comprising a nucleotide sequence identical or equivalent to at least a part of the nucleotide sequence of a fragment defined in Claim 7, in particular a nucleotide sequence having, for any sequence of 10 contiguous monomers, at least 70% 30 homology with at least a part of the said fragment, in order to amplify, by polymerization, an SNA or a DNA of a viral material associated with rheumatoid arthritis.

9. Use according to Claim 8, characterized in that the primer comprises a nucleotide sequence chosen from 35 LitSQ ID N016, SEQ ID N017, SEQ ID N018, SEQ ID N019, SEQ XD N020, SEQ ID N021, SEQ ID N022, SEQ ID N023, SEQ ID N024, SEQ ID N025, SEQ ID N026, SEQ ID N031, SEQ ID N032, SEQ ID N033, SEQ ID N047, SEQ ID N048 and SEQ ID N049 and complementary sequences thereof. • -»- 3 0 3 6 9 4

10. Use of a probe comprising a nucleotide sequence identical or equivalent to at least a part of the nucleotide sequence of a fragment defined in Claim 6, in particular a nucleotide sequence having, for any sequence 5 of 10 contiguous monomers, at least 70% homology with at least a ^art of the said fragment, in order to obtain a composition to detect, separate or identify, in a biological sample, a viral material associated with rheumatoid arthritis. 10

11. Use according to Claim 10, characterized in that the probe comprises a nucleotide sequence chosen from SEQ ID N03, SEQ ID N04, SEQ ID N05, SEQ ID N06, SEQ ID N07, SEQ ID NOl 6, SEQ ID N017, SEQ ID N018, SEQ ID N019, SEQ ID N020, SEQ ID N021, SEQ ID N022, SEQ ID N023, SEQ ID 15 N024, SEQ ID N025, SEQ ID N026, SEQ ID N031, SEQ ID N032, SEQ ID N033, SEQ ID N047, SEQ ID N048 and SEQ ID N049 and complementary sequences thereof.

12. Use of a pathogenic and/or infective agent, in the purified or isolated state, different from the viral material defined in any one of Claims 1 to 6, as derived from a viral strain chosen from the strains referred to respectively as POL-2, filed on 22.07.1992 with the ECACC under the access number V92072202, and HS7P6 filed on 08.01.93 with the ECACC under the access number V93010816, and from the variant strains thereof, consisting of pathogenic emd/or infective agents comprising at least one antigen which is recognized by at least one antibody directed against at least one antigen corresponding to either of the pathogenic and/or infective agents of the abovementioned viral strains POL-2 and MS7PG, which are respectively different from either retroviral material of the said strains, in order to obtain a diagnostic, prophylactic or therapeutic composition for detecting, preventing or treating an infection by the said pathogenic and/or infective agent associated with rheumatoid arthritis, or a reactivation of the said agent.

13. Use of a pathogenic and/or infective agent, in the purified or isolated state, different from the v.frel 20 i » • f / I / 30 35 303694 - 84 - material defined in any one of Claims 1 to 6, as produced by a cell line chosen from the lines referred to respectively as PLI-2 fi.l*d on 22.07.1992 with the ECACC under the access number 92072201, and LM7PC filed on 08.01.93 5 with the ECACC under the access number 93010817, and by any infected cell cultures capable of producing at least either of the pathogenic and/or infective agents, and/or variants thereof, or by any infected cell culture capable of producing a pathogenic and/or infective agent compris-10 ing at least one antigen which is recognized by at least one antibody directed against at least one antigen corresponding to either of the pathogenic and/or infective agents produced by the abovementioned lines PLI-2 and LM7PC, in order to obtain a diagnostic, prophy-15 lactic or therapeutic composition for detecting, preventing or treating an infection by the said pathogenic and/or infective agent associated with rheumatoid arthritis, or a reactivation of the said agent.;

14. Use of a pathogenic and/or infective agent 20 comprising a nucleic acid comprising a nucleotide sequence chosen from SEQ ID NOlO, SEQ ID NOll, SEQ ID N012, SEQ ID N040, SEQ ID N041, complementary sequences thereof and equivalent sequences thereof, in particular the nucleotide sequences having at least 70% 25 and preferably at least 90% homology with a nucleotide sequence comprising a sequence chosen from SEQ ID NOlO, SEQ ID NOll, SEQ ID N012, SEQ ID N040, SEQ ID N041 and complementary sequences thereof, in order to obtain a diagnostic, prophylactic or therapeutic composition for 30 detecting, preventing or treating an infection by the said pathogenic and/or infective agent associated with rheumatoid arthritis, or a reactivation of the said agent.;

15. Use of a nucleotide fragment comprising a nucleotide 35 sequence chosen from SEQ ID NOlO, SEQ ID NOll, SEQ ID;N012, SEQ ID N040, SEQ ID N041, complementary sequences thereof and equivalent sequences thereof, in particular the nucleotide sequences having, for any sequence of 100 contiguous monomers, at least 70% and preferably at least;- 85 -;3036 94;90% homology with a sequence chosen from SEQ ID NOlO, SEQ ID NOll# SEQ ID N012, SEQ ID N040 and SEQ ID N041 and complementary sequences thereof, in order to obtain a diagnostic, prophylactic or therapeutic composition for 5 detecting, preventing or treating an infection by the said pathogenic and/or infective agent associated with rheumatoid arthritis, or a reactivation of the said agent.;

16. Use of a primer comprising a nucleotide sequence 10 identical or equivalent to at least a part of the nucleotide sequence of a fragment defined in Claim 15, in particular a nucleotide sequence having, for any sequence of 10 contiguous monomers, at least 90% homology with at least a part of the said fragment, in order to amplify by 15 polymerization an SNA or a DNA of a pathogenic and/or infective agent associated with rheumatoid arthritis.;

17. Use according to Claim 16, characterized in that the primer comprises a nucleotide sequence choBen from SEQ ID N013, SEQ ID N014, SEQ ID N015, SEQ ID N027, SEQ;20 ID N028, SEQ ID N029, SEQ ID N030, SEQ ID N034, SEQ ID N035, SEQ ID N036, SEQ ID N037, SEQ ID N044, SEQ ID N045 and SEf ID N046 and complementary sequences thereof.;

18. Use of a probe comprising a nucleotide sequence identical or equivalent to at least a part of the;25 nucleotide sequence of a fragment according to Claim 15, in particular a nucleotide sequence having, for any sequence of 10 contiguous monomers, at least 90% homology with at least a part of the said fragment, iu order to obtain a diagnostic, prophylactic or therapeutic 30 composition for detecting, preventing or treating em infection by the said pathogenic emd/or infective agent associated with rheumatoid arthritis, or a reactivation of the said agent.;

19. Use according to Claim 18, characterized in that 35 the probe comprises a nucleotide sequence chosen from SEQ;ID NOlO, SEQ ID NOll, SEQ ID N013, SEQ ID N014, SEQ ID N015, SEQ ID N027, SEQ ID N028, SEQ ID N029, SEQ ID N030, SEQ ID N034, SEQ ID N035, SEQ ID N036, SEQ ID N037, SEQ ID N044, SEQ ID N045 and SEQ ID N046 and comple-;- 86 -;30 3 6 9 4;mentary sequences thereof.;

20. Use of an association comprising two pathogenic and/or infective agents, in the isolated or purified state, namely, a first agent which consists of a human;5 virus, possessing reverse transcriptase activity, and related to a family of endogenous retroviral elements, or a variant of the said virus, and a second agent, or a variant of the said second agent, these two pathogenic and/or infective agents being as those derived from the 10 same viral strain chosen from the strains referred to respectively as POL-2, filed on 22.07.1992 with the ECACC xmder the access number V92072202, and MS7PG filed on 08.01.93 with the ECACC under the access number V93010816, and from the variant strains thereof, in order 15 to obtain a diagnostic, prophylactic or therapeutic composition for detecting, preventing or treating an infection by a first pathogenic and/or infective agent, and a second pathogenic and/or infective agent, associated with rheumatoid arthritis, or a reactivation of 20 either the said first agent or the said second agent.;

21. Use of an association comprising two pathogenic and/or infective agents, in the isolated or purified state, namely, a first agent consisting of a human virus possessing reverse transcriptase activity, and related to;25 a family of endogenous retroviral elements, or a variant of the said virus, and a second agent, or a variant of the said second agent, these two pathogenic and/or infective agents being as those produced by the same cell line chosen from the lines referred to respectively as 30 PLI-2, filed on 22.07.1992 with the ECACC under the access number 92072201, and LM7PC, filed on 08.01.93 with the ECACC under the access number 93010817, and by any infected cell cultures capable of producing at least either of the pathogenic and/or infective agents, and/or 35 variants thereof, in order to obtain a diagnostic, prophylactic or therapeutic composition for detecting, preventing or treating an infection by a first pathogenic and infective agent, and by a second pathogenic and/or infective agent, associated with rheumatoid arthritis, or;• 303694;a reactivation of either the said first agent or the said second agent.;

22. Use of an association comprising two pathogenic and/or infective agents, in the isolated or purified 5 state, namely a first agent consisting of a virus, or of a variant oi *-.he said virus, the genome of wloich comprises a nucleoti.de sequence chosen from SEQ ZD NOl, SEQ ID N02, SEQ ID N03, SEQ ID N04, SEQ ID NOS, SEQ ID N06, SEQ ID N07, SEQ ID N08, SEQ ID N09, SEQ ID N039, SEQ ID 10 N042 and SEQ ID N043, complementary sequences thereof and equivalent sequences thereof, in particular the nucleotide sequences having, for any sequence of 100 contiguous monomers, at least 50% and preferably at least 70% homology with a nucleotide sequence chosen from 15 SEQ ID NOl, SEQ ID N02, SEQ ID N03, SEQ ID N04, SEQ ID N05, SEQ ID N06, SEQ ID N07, SEQ ID N08, SEQ ID N09, SEQ ID NO.'}9, SEQ ID N042 and SEQ ID N043 and complementary sequences thereof,, and a second pathogenic and/or infective agent, the genome of which comprises a 20 nucleotide sequence chosen from SEQ ID NOlO, SEQ ID NOll, and SEQ ID N012, SEQ ID N040 and SEQ ID N041, complementary sequences thereof and equivalent sequences thereof, in particular the nucleotide sequences having, for any sequence of 100 contiguous monomers, at least 70% and 25 preferably at least 90% homology with a nucleotide sequence chosen from SEQ ID NOlO, SEQ ID NOll, SEQ ID N012, SEQ ID N040 and SEQ ID N041, and complementary sequences thereof, in order to obtain a diagnostic, prophylactic or therapeutic composition for detect-30 ing, preventing or treating an infection by a first pathogenic and/or infective agent, and by a second pathogenic and/or infective agent, associated with rheumatoid arthritis, or a reactivation of either the said first agent or the said second agent. 35

23. Use of an association of nucleotide fragments comprising a first fragment whose nucleotide sequence comprises a nucleotide sequence chosen from SEQ ID NOl, SEQ ID N02, SEQ ID N03, SEQ ID N04, SEQ ID N05, SEQ ID N06, SEQ ID N07, SEQ ID N08, SEQ ID N09, 3 0 3 6 9 4 - 88 - SEQ ID N039, SEQ ID N042 and SEQ ID N043, complementary sequences thereof and equivalent sequences thereof, In particular the nucleotide) sequences having, for any sequence of 100 contiguous monomers, at least 50% and 5 preferably at least 70% homology with a nucleotide sequence chosen from SEQ ID NOl, SEQ ID N02, SEQ ID N03, SEQ ID N04, SEQ ID N05, SEQ ID N06, SEQ ID N07, SEQ ID N08, SEQ ID N09, SEQ ID N039, SEQ ID N042 and SEQ ID N043 and complementary sequences thereof, and a 10 second fragment whose nucleotide sequence comprises a nucleotide sequence chosun from SEQ ID NOlO, SEQ ID NOll, SEQ ID N012, SEQ ID N040 and SEQ ID N041, complementary sequences thereof and equivalent sequences thereof, in particular the nucleotide sequences having, for any 15 sequence of 100 contiguous monomers, at least 70% and preferably at least 90% homology with a nucleotide sequence chosen from SEQ ID NOlO, SEQ ID NOll, SEQ. ID N012, SEQ ID N040 and SEQ ID N041 and complementary sequences thereof, each of the said fragments being in 20 particular a probe, in order to obtain a diagnostic, prophylactic or therapeutic composition for detecting, preventing or treating an infection by a first pathogenic and/or infective agent, and a second pathogenic and/or infective agent, associated with rheumatoid arthritis, or 25 a reactivation of either the said first agent or the said second agent.

24. Use of an association comprising a first polypeptide partially or totally coded for by the first nucleotide fragment defined in Claim 23, and a second 3 0 polypeptide partially or totally coded for by the second nucleotide fragment defined in Claim 23, in order to obtain a diagnostic, prophylactic or therapeutic composition for detecting, preventing or treating an infection by a first pathogenic and/or infective agent, and a 35 second pathogenic and/or infective agent, associated with rheumatoid arthritis, or a reactivation of either the said first agent or the said second agent.

25. Use according to Claim 24, characterized in that the use comprises a first ligand, in particular an anti- - 89 - 30 3 6 9 4 body, which is specific for the first polypeptide, and a second ligand, in particular an antibody, which is specific for the second polypeptide, the said first and second polypeptides being defined in Claim 24. 5

26. Process for the production of a first pathogenic and/or infective agent according to one of Claims 1 to 6 and/or of a second pathogenic and/or infective agent according to either of Claims 13 and 15, associated with rheumatoid polyarthritis, characterized in that cells 10 from synovial fluid punctures are cultured in vitro, these cells being chosen in particular from the desquamated fibroblasts and synoviocytes of articular fluid, from patients suffering from rheumatoid arthritis.

27. Process for the production of a first pathogenic 15 and/or infective agent according to one of Claims 1 to 6 and/or of a second pathogenic and/or infective agent according to either of Claims 13 and 14, associated with rheumatoid arthritis, characterized in that B lymphocyte cells immortalized by Epstein-Barr virus are cultured in 20 vitro, these cells coming from patients suffering from rheumatoid arthritis.

28. Nucleotide fragment whose nucleotide sequence comprises a sequence chosen from SEQ XD N039, SEQ XD N042 and SEQ XD N043, complementary sequences thereof and 25 equivalent sequences thereof, in particular the nucleotide sequences having, for any sequence of 100 contiguous monomers, at least 50% and preferably at least 70% homology with a sequence chosen from SEQ XD N039, SEQ XD N042 and SEQ ID N043 and complementary sequences 3 0 thereof.

29. Specific primer for the amplif ication by polymerization of an SNA or a DNA of a viral material according to any one of Claims 1 to 6, characterized in that it comprises a nucleotide sequence identical or 35 equivalent to at least a part of the nucleotide sequence of a fragment according to Claim' 28, in particular a nucleotide sequence having, for any sequence of 10 contiguous monomers, at least 70% homology with at least a part of the said fragment. - 90 - 30 3 6 94

30. Primer according to Claim 29, characterized in that it comprises a nucleotide sequence chosen from SEQ ID N047, SEQ ID N048 and SEQ ID N049 and complementary sequences thereof. 5

31. Probe capable of specifically hybridizing with an RNA or a DNA of a viral material according to any one of Claims 1 to 6, characterized in that it comprises a nucleotide sequence identical or equivalent to at least a part of the nucleotide sequence of a fragment according 10 to Claim 28, in particular a nucleotide sequence having, for any sequence of 10 contiguous monomers, at least 70% homology with at least a part of the said fragment.

32. Probe according to Claim 31, characterized in • that it comprises a nucleotide sequence chosen from 15 SEQ ID N047, SEQ ID N048 and SEQ ID N049 and complementary sequences thereof.

33. Nucleotide fragment whose nucleotide sequence comprises a sequence chosen from SEQ ID N040 and SEQ ID N041, complementary sequences thereof and equiva- 20 lent sequences thereof, in particular the nucleotide sequences having, for any sequence of 100 contiguous monomers, at least 70% and preferably at least 90% homology with a sequence chosen from SEQ ID N040 and SEQ ID N041 and complementary sequences thereof. 25

34. Specific primer for the amplif ication by polymerization of an RNA or a DNA of a pathogenic and/or infective agent according to one of Claims 12 to 14, comprising a nucleotide sequence identical or equivalent to at least a part of the nucleotide sequence of a 30 fragment according to Claim 33, in particular a nucleotide sequence having, for any sequence of 10 contiguous monomers, at least 90% homology with at least a part of the said fragment.

35. Primer according to Claim 34, characterized in 35 that it comprises a nucleotide sequence chosen from SEQ ID N044, SEQ ID N045 and SEQ ID N046 and complementary sequences thereof.

36. Probe capable of specifically hybridizing with an RNA or a DNA of a pathogenic and/or infective agent 91 30 36 94 according to one of Claims 12 to 14, comprising a nucleotide sequence identical or equivalent to at least a part of the nucleotide sequence of a fragment according to Claim 33, in particular a nucleotide sequence having, for any sequence of 10 contiguous monomers, at least 90% homology with at least a part of the said fragment.

37. Probe according to Clai'a 36, characterized in that it comprises a nucleotide sequence chosen from SEQ ID N044, SEQ ID N045 and SEQ ID N046 and complementary sequences thereof. END OF CLAIMS