WO1989009815A1 - Novel virus of the hiv-2 family and methods of detection therefor - Google Patents

Abstract

The present invention relates to a novel virus, said virus belonging to the HIV-2 family of viruses. In particular, the invention relates to a retrovirus characterized by being antigenically more similar to, but genetically distinct from, HIV-2/ROD and SIVMAC than to HIV-1. More particularly, the present invention relates to the isolation and cultivation of HIV-2/ST thereby providing a source of said virus and its derivatives including virus-derived antigens and derivatives and parts thereof, useful in the development of diagnostic assays for said virus or other related viruses belonging to the HIV-2 family. Furthermore, the present invention is also directed to recombinant DNA molecules containing the entire HIV-2/ST provirus thereby providing a source of recombinant viral components useful in the development of said diagnostic assays for HIV-2 viruses. The present invention is also directed to antibodies specific to viral components and to different antibodies specific to the first antibodies, said antibodies are also useful in the development of diagnostic assays for HIV-2 viruses. The present invention also contemplates a method of the isolation of HIV-2/ST-type viruses.

Description

NOVEL VIRUS OF THE HIV-2 FAMILY AND METHODS **■_ OF DETECTION THEREFOR

FIELD OF THE INVENTION

The present invention relates to" a novel virus, said virus belonging to the HIV-2 family of viruses. In 5 particular, the invention relates to a retrovirus characterized by being antigenically more similar to

HIV-2/ROD and SIV M..A-C- than to HIV-1. More p ^carticularly^J, the present invention relates to the isolation and cultivation of HIV-2/ST thereby providing a source of said virus and its

-"LO derivatives, including virus-derived antigens and derivatives and parts thereof, useful in the development of diagnostic assays for said virus or other related viruses belonging to the HIV-2 family. Furthermore, the present invention is also directed to recombinant DNA molecules containing the entire

^*-*_-*-*,^' HIV-2/ST provirus thereby providing a source of recombinant viral components useful in the development of said diagnostic assays for HIV-2 viruses. The present invention is also directed to antibodies specific to viral components and to second antibodies specific to the first antibodies, said

2o second antibodies are also useful in the development of diagnostic assays for HIV-2 viruses. The present invention also contemplates a method for the isolation of HIV-2/ST-type viruses. BACKGROUND OF THE INVENTION

25 Acquired immune deficiency syndrome (AIDS) has been etiologically linked to infection by a human retrovirus, variously designated lymphadenopathy-AIDS-virus (LAV) , human T-lymphotropic virus type III (HTLV-III) , AIDS-related virus (ARV) and human immunodeficiency virus type I (HIV-1) .

-DQ Infection with HIV-1 is recognized as an epidemic of global dimensions with Africa evidencing the highest prevalence.

35 Following the detection of HIV-1 as the causative agent for

AIDS it was discovered that the T-lymphotrophic retrovirus family not only included HIV-1 and bovine leukemia virus but also closely related agents that infect certain non-human primate species. For example, simian T-lymphotropic virus type-I (STLV-I) naturally infects most species of so called

Old World monkeys and great apes. STLV-I immortalizes T lymphocytes Ln vitro, similar to its human counter part HIV-1, and has been linked with spontaneous lymphoid malignancy in the primate host. Another simian

T-lymphotropic virus, SIV, has been described in captive ill rhesus macaques (Macaca mulatta) and healthy wild-caught

African Green monkeys (Cercopithecus sp.) (Kanki, Alroy and

Essex Science 230:951-954, 1985) . This virus has a cytolytic effect on T4 lymphocytes in culture, Mg 2+-dependent reverse transcriptase and retroviral particles with morphology similar to HIV-1.

Subsequent to the identification of SIV, a novel human retrovirus was isolated from two AIDS patients from

West Africa (Clavel et al. Science 233;343-346, 1986) . This virus has been designated LAV-II or HIV-2/R0D; the latter designation is adopted herein. The virus HIV-2/R0D has biological and morphological properties very similar to HIV-1 but differs in some of its antigenic components and differs substantially in envelope glycoproteins. Interestingly, the envelope antigen of HIV-2/R0D can be recognized by serum from a macaque with simian AIDS (i.e. infected with SIV) therefore suggesting that HIV-2/R0D may be more closely related to SIV than to HIV-1. Hybridization experiments with HIV-1 subgenomic probes established that HIV-2/ROD is distantly related to HIV-1 and distinct from SIV. Serologic evidence was used to detect another viral isolate similar to SIV infecting healthy people in Senegal,

West Africa, which was designated HTLV-IV .(Kanki e_t al.

Science 232:238-243, 1986) . This virus was shown to have retroviral type particles, growth characteristics and major viral proteins similar to those of the SIV and HIV-1 group of viruses.

Because of similarities in structure and antigenicity among HIV-1, HIV-2/R0D and SIV, some relation might be expected between their nucleic acid sequences and phylogenesis. Restriction enzyme analysis showed that six out of six SIV_AG„ (African Green Monkey) isolates and two out of three HTLV-IV isolates were identical in 32 out of 32 restriction sites mapped (Hirsch e_t _al. , P.N.A.S. USA 83:9754-9758, 1986; Kornfeld et al. , Nature 326:610-613, 1987; Hirsch et al. Cell 49:307-319, 1987) . The third HTLV-IV isolate was identical to the others in 31 out of 32 mapped restriction sites. Of three SIV_MAC (Macaque) isolates, one was identical to the SIV _,/HTLV-IV consensus pattern in 23 out of 23 sites and the others were identical to each other and similar to the consensus pattern in 21 out of 23 sites (Kornfeld e_t a_l. , supra) . The same genetic analysis clearly distinguished HIV-2 from these viruses (Guyader et al. Nature 326:662-669, 1987) .

To define more precisely the relation between different HTLV-IV, SIV, HIV-2 and HIV-1 viruses, Hahn et al. (Nature 330:184-186, 1987) used a combination of restriction endonuclease cleavage mapping, molecular cloning and nucleotide sequencing to analyze and compare different isolates of HTLV-IV and SIV. This analysis showed that the HTLV-IV reported by Kanki e_t al. supra and the SIV _M reported by Kanki, Alroy and Essex supra were not independent virus isolates but instead represent transmission of a single laboratory virus strain to multiple cultures. Furthermore, the various HTLV-IV and SIV_A isolates probably originated from cultures of SIV M,,A_Λ„C (isolate 251) since the

HTLV-IV/SIV _G restriction site consensus pattern coincided precisely with isolate 251 in 32 out of 32 mapped sites but is similar to SIV - isolate 142 in only 27 out of 31 sites

(Kornfeld et al. , supra; Chakrabarti et _al. Nature 328:543-547, 1987; Desrosiers et al. Nature 327:107, 1987) .

On the basis of clinical (Clavel et al. New Ξngl.

J. Med. 316:1180-1185, 1987) and seroepidemiologic (Kanki et al. Science 226:1165-1171, 1984) studies of West African populations, it is clear that a virus or family of viruses exists which is antigenically more similar to HIV-2/R0D and ^SIV _MAC ^{tiιan to}. ^HIV~ - ^an3- is prevalent and in some case associated with immunodeficiency. There is clearly a need, therefore, for a method to isolate these viruses, and, once accomplished, to use these viruses to develop diagnostic assays to detect same and related viruses. The present invention fulfils this need. SUMMARY OF THE INVENTION

The present invention relates to a novel virus, said virus isolated from human subjects seropositive for the HIV-2/R0D virus, which is characterized by being antigenically more similar to, but genetically distinct from, HIV-2/R0D and SI _MAC than to HIV-1. The virus is further characterized by being a retrovirus. More particularly, said virus has the identificable characteristics of HIV-2/ST.

The present invention also contemplates a method of isolating said virus and its culturing in ^"immortalized T-lymphocytes. Another aspect of this invention is directed to recombinant DNA molecules containing the entire HIV-2/ST provirus thereby providing a source of recombinant viral components useful in the development of diagnostic assays for

HIV-2 viruses.

Yet, another aspect of this invention relates to the use of said virus, its derivatives and parts thereof,

(e.g. protein coat) or recombinant viral components, or derivatives or parts thereof, in the development of diagnostic assays for HIV-2/ST and antigenically related viruses, such as HIV-2/R0D and SIV.

Still another aspect of this invention is the use of said virus, or derivatives or parts thereof (e.g. protein coat) or recombinant viral components, or derivatives or parts thereof, to generate antibodies useful in diagnostic and therapeutic techniques. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a photographic representation of HIV-2/ST viral particles.

Figure 2 is a photographic representation of a Southern blot hybridization showing the relative nucleotide sequence homology of HIV-2/ST to HIV-2/R0D (panel a) and SIV (HTLV-IV) (panel b) . Lane 1 contains HTLV-IV/Pk82 of Kanki ej aJL. supra; Lane 2 contains normal human DNA; Lanes 3-5 contain HIV-2/ST from Hut78 cells; Lane 6 contains HIV-2/ST from SupTl cells.

Figure 3 is a photographic representation of Southern blot hybridization and restriction endonuclease digestion of HIV-2/ST.

Figure 4 is a photographic representation of a Western blot (panel A) and radioimmunoprecipitation (panels B and C) analysis of HIV-2/ST, HIV-2/R0D, SIV_MAC and HIV-1/IIIb proteins. Figure 5 is a photographic representation showing syncytium formation by HIV-2/ST, HIV-2/R0D and HIV-1/IIIb infected cells and CD4 positive SupTl (panel A) and Hela-T4 (panel B) indicator cells.

Figure 6 is a graphical representation of a comparison of cytophatic/cell killing properties of HIV-2/ST and prototype HIV-1 (isolates Illb, BC, WMJ, RH) and HIV-2 (isolate ROD) viruses.

Figure 7 is a photographic representation depicting an in situ hybridization analysis of cell-free viral infection by HIV-1/IIIb, HIV-2/R0D and HIV-2/ST.

Figure 8 is a graphical representation of the level of reverse transcriptase in T-lymphocytes infected with HIV-2/ST.

Figure 9 is a representation of the cDNA sequence of HIV-2/ST-type virus. DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, a new and novel virus has been isolated from human subjects seropositive for the HIV-2/R0D virus. This virus is designated herein HIV-2/ST and has the characteristics of a retrovirus and being antigenically more similar to, but genetically distinct from, HIV-2/R0D and SIV than to HIV-1. As defined herein, antigenically more similar refers to a determination based on iramunoreactivity using HIV-2/R0D antibodies and genetic relatedness is measured by nucleic acid analysis (e.g. southern blot, restriction analysis) . Moreover, the relationship of HIV-2/ST relative to HIV-2/R0D and SIV_MAC is further shown by analysis of ultrastruetural morphology, particulate reverse transcriptase activity, T. cell tropism and antigenic reactivity by Western blot analysis. The identification, isolation and culturing of HIV-2/ST provides a heretofor unavailable source of virus particles and parts thereof, and antigenic determinants and parts thereof, being highly desirable for their medical and experimental utility.

Thus, among the many advantages of the present invention, it has been surprisingly discovered that c HIV-2/ST is more related to HIV-2/R0D and SIVM„A-C_ than to

HIV-1 and isolates previously designated as HTLV-IV. Since previous HTLV-IV isolates in fact belong to the SIV family

(Hahn e_t a_l. supra) , these viruses will henceforth be .designated SIV (HTLV-IV) . Southern blot analysis of cellular DNA isolated from immortalized T-lymphocytes infected with HIV-2/ST using a specific HIV-2 DNA probe and an SIV

(HTLV-IV) probe (see Example 2) clearly establish that HIV-2/ST is more related to HIV-2/R0D than to SIV(HTLV-IV) .

It is considered within the scope of the present invention, all viruses exhibiting one or more of the aforementioned characteristics including, for example, the virus HIV-2/ST and its derivatives and the individual and collective components, structural or otherwise, contained therein. The RNA and associated reverse transcriptase contained in the HIV-2/ST virus as well as the corresponding DNA from said RNA and any recombinant molecules generated therefrom are also contemplated to be within the scope of the present invention.

In accordance with the subject invention, three full length HIV-2/ST proviral clones were obtained from an immortalized T-lymphoσyte line infected with the virus. The infected cell line is designated Hut78/Bl2 and the clones obtained are designated JSP4-27, JSP4-32 and JSP4-34. Total cellular DNA from Hut78/B12 was isolated using standard techniques, subjected to Sau3A partial digestion and cloned into BamHl-digested Jl-lambda phage. A cos id library was thus obtained in Escherichia coli. Clones carrying HIV-2/ST proviral DNA were identified by plaque hybridization using a full-length SIV probe (see Hahn et al. , supra) . The three aforementioned clones were thus identified. Techniques useful in cloning the HIV-2/ST proviral DNA can be found in Maniatis e_t a_l. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982, pp. 1-500 and in Shaw e_t al. Science 266:1165-1171, 1984. These clones provide a source of recombinant viral components when ligated into suitable expression vectors.

Genetic analysis of HIV-2/ST in comparison with prototype strains of HIV-1, HIV-2/ROD and SIV was conducted (Example 3) . The DNA genome of the HIV-2/ST virus hybridized at low and high stringency to the prototype HIV-2/ROD clone whereas SI hybridized to the same HIV-2/ROD probe only under conditions of low stringency. HIV-1 did not hybridize to the HIV-2/R0D probe under any conditions. Conversely, clones of SIV_MAC used as probe hybridized to HIV-2/ROD and HIV-2/ST with equal intensity but only under conditions of low stringency. A full-length HTLV-1 probe gave no hybridization. Two different HIV-2/ST producing cell lines derived from the transmission of cell-free virus from primary ST lymphocyte co-cultures exhibited very similar proviral restriction enzyme cleavage patterns but were distinguishable by polymorphic Xbal and BamHl sites. Comparative restriction mapping of the three full-length HIV-2/ST proviral clones confirmed the genetic similarity between HIV-2/ST and HIV-2/ROD (10 of 25 restriction sites in common) and the dissimilarity between HIV-2/ST and SIV,^-, (2 of 36 restriction sites in common) . Furthermore, one of these proviral DNA clones (lambda JSP-27) _r when transfected into T-lymphocyte cell lines. produced virus that was replication-competent and infectious and whose biological properties were identical to the parental virus isolate. Together, these studies showed conclusively that the HIV-2/ST virus is genotypically more related to prototype HIV-2/R0D than to other primate viruses, that within the HIV-2/ST virus isolate there exist polymorphic replication competent viral genotypes, and that an SIV-like virus is not present in the HIV-2/ST cultures.

The structural, antigenic, and functional characteristics of HIV-2/ST proteins were evaluated by

Western blot and radioimmunoprecipitation (RIP) analyses

(Example 4) . The putative major gag structural proten and precursor (p26/pr55) of HIV-2/ST were identified and were comparable in size to p26/pr55 of HIV-2/R0D, p27/pr55 of

SIV _,, and p24/pr53 of HIV-1. Polymerase and endonuclease proteins of HIV-2/ST were tentatively identified as p64 and p34 proteins with equivalent counterparts in HIV-2/ROD,

SIVM„A_ΛC_,, and HIV-1. The envelope proteins of HIV-2/ST revealed an approximately 140 kilodalton (kD) putative extracellular protein similar to that in HIV-2/ROD. However, in different subcultures of HIV-2/ST that were derived by single cell cloning of the original SupTl/LKOOl cell line, the putative transmembrane proteins (TMP) were either 42 kD or 30 kD in size. Only smaller-sized transmembrane proteins were seen in HIV-2/R0D (36 kD) and SIV_MAC (32 kD) cultures. The HIV-2/ST viruses possessing large and small transmembrane proteins (TMP) could be transmitted cell free, were replication competent, and retained the same size of TMP throughout passage in the same cell targets (SupTl and CEM x 174) . This argued that the observed differences in TMP sizes were due to nucleotide sequence differences in the expressed mRNAs and were not due to post-transcriptional modifications such as altered glyσosylation patterns. Antigentically, HIV-2/ST and HIV-2/R0D were similar to each other and to SI in their reactivity with HIV-2/R0D antisera and they were cross-reactive with HIV-1 only in the major gag structural proteins (p24-p27) . This was confirmed by bi-directional immunoblotting in which both

HIV-1 and HIV-2 specific human antisera were used to probe

HIV-1, HIV-2/R0D and HIV-2/ST proteins. Radioimmuno¬ precipitation demonstrated envelope precursor proteins of approximately 180 or 170 kD size in the different HIV-2/ST subcultures reflecting the different sizes of their respective transmembrane proteins, whereas the mature extracellular envelope proteins were equal in size (140 kD) . RIP analysis also demonstrated direct binding of the HIV-2/ST external envelope glycoprotein to an epitope on T4(CD4) recognized by 0KT4A antibodies, but only by OKT4 antibodies, similar to the binding specificity of HIV-1. Other experiments using fluorescence activated cell sorting analysis demonstrated that infection of T4(CD4) bearing cells by HIV-2/ST virus down-modulated the expression of T4 but not T8, again analogous with HIV-1 infection.

Because of serological and clinical evidence suggesting that certain West African human populations infected with HIV-2 related viruses may have less severe immunodeficiency than do individuals infected with HIV-1, or some isolates of HIV-2 including HIV-2/ROD the HIV-2/ST virus was evaluated for cytopathic and cell killing properties in vitro. Cytopathic properties were first studied by examining the induction of syncytia by HIV-2/ST infected cells co-cultured with a panel of four different T4 (CD4)-bearing indicator cells (SupTl, Hela-T4, H9, Hut78) . Figure 5 shows syncytia formation by prototype HIV-1/IIIb and HIV-2/ROD infected cells in comparison with HIV-2/ST infected cells using Sup Tl and HeLa-T4 cells as indicator lines. Both HIV-1/IIIb and HIV-2/ROD reproducibly produced large syncytia whereas HIV-2/ST failed to do this. T4 (CD4) bearing Hut 78 and H9 indicator cells also developed large syncytia when co-cultivated with HIV-1/HTLVIIIb and HIV-2/R0D infected cells whereas HIV-2/ST produced no syncytia. Syncytia- induction was completely inhibited by anti-Leu3a antisera and no syncytia were formed by any HIV-1 or HIV-2 infected cells when co-cultured with indicator cells lacking T4 (CD4) .

Cytopathicity was also assessed by comparing the cell killing properties of cell-free, infectious HIV-2/ST with different isolates of HIV-1 and HIV-2/R0D against variety of different target cells (Figure 6) . HIV-2/R0D and two different isolates of HIV-1 (Illb and BC) caused marked cell killing of

SupTl cells (panels A and B) , PHA-stimulated normal donor lymphocytes (panels C and D) , as well as Hut78 and H9 cells

(not shown) . In contrast, equivalent amounts of HIV-2/ST showed no detectable cell killing activity against the same cell targets. Even 10- and 100-fold more HIV-2/ST virus than three other HIV-1 viruses (WMJ, RH, BC) led to only transient slight depression in cell counts (Panel E) .

The present invention also contemplates a method for the isolation and culturing of the viruses considered herein. Henceforth, said viruses are designated HIV-2/ST-type viruses .of which HIV-2/ST is an example thereof. The isolation procedure, outlined in more detail in Example 1, utilizes repeated polyethylene glycol (PEG) mediated virus precipitations from culture supernatant fluid which are in turn incubated repeatedly with the same T-lymphocytes.

It is within the scope of this invention to include . any T-lymphocyte population infected with HIV-2/ST, but preferably T4-enriched populations, including the lines Hut78, SupTl, CEMX174 and H9. In a preferred embodiment, immortalized T-lymphocyte lines Hut78, SupTl and CEMX174 are highly infected by HTLV-IV/ST and the corresponding cultures are designated Hut78/B12, SupTl/LKOOl and CEMX174/ST, respectively. These cultures provide a convenient and prolific source of the heretofor not discovered HIV-2/ST.

Furthermore, in accordance with the subject invention, Hut78/Bl2, SupTl/LKO01 and CEMX174/ST provide a stable, permanent source of virus for diagnostic testing of patient sera and a stable, permanent source of virus and viral nucleic acid sequences (RNA and DNA) for the production of recombinant DNA based diagnostic reagents, vaccines and therapies for AIDS and AIDS-related diseases.

One skilled in the art will immediately recognize the utility of HIV-2/ST discovered in accordance with this invention. It is known that HIV-2/ST or similar viruses

(e.g. HIV-2/R0D) are associated with some forms of, and to certain degrees of, immunodeficiency in human subjects.

Furthermore, HIV-2/R0D is similarly an etiological agent of an immunodeficiency disease. In accordance with the subject invention, HIV-2/ST can be used to isolate antigenic or genetic determinants useful in screening blood samples for the presence of HIV-2/ST and HIV-2-type viruses.

For example, the present invention contemplates the use of structural components such as the major structural protein of the virus core, the external envelope protein, the envelop transmembrane protein, the lipid membrane and the outer core protein, singularly or in combination, and isolated from HIV-2/ST, to prepare antibodies. Said antibodies may be monoclonal or polyclonal. Additionally, it is within the scope of this invention to include second antibodies (monoclonal or polyclonal) directed to the first antibodies discussed above. The present invention further contemplates use of these antibodies in a detection assay (immunoassay) for HIV-2/R0D and HIV-2/ST type viruses.

HIV-2/ST and its antigenic components can be purified then utilized in antibody production. Both polyclonal and monoclonal antibodies are obtainable by immunization with the virus or its components, and either type is utilizable for immunoassays. The methods of obtaining both types of sera are well known in the art.

Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of the purified virus or antigenic component, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques.

Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favored because of the potential heterogeneity of the product.

The use of monoclonal antibodies in the present immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art. (See, for example,

Douillard, J. Y. and Hoffman, T., "Basic Facts About

Hybridomas", in Compendium of Immunology Vol. II, L. Schwartz

(Ed.) (1981) ; Kohler, G. and Milstein, C, Nature 256:495-497

(1975) ; European Journal of Immunology, Vol. 6, pp. 511-519 (1976, Koprowski et al . , U.S. Patent 4,172,124, Koprowski et al. , U.S. Patent 4,196,265 and Wands, U.S. Patent 4,271,145, the teachings of which are herein incorporated by reference.) Unlike preparation of polyclonal sera, the choice of animal is dependent on the availability of appropriate immortal lines capable of fusing with lymphocytes thereof. Mouse and rat have been the animals of choice in hybridoma technology and are preferably used. Humans can also be utilized as sources for sensitized lymphocytes if appropriate immortalized human (or nonhuman) cell lines are available.

For the purpose of the present invention, the animal of choice may be injected with from about 1 mg to about 20 mg of the purified virus or antigenic component thereof. Usually the injecting material is emulsified in Freund's complete adjuvant. Boosting injections may also be required. The detection of antibody production can be carried out by testing the antisera with appropriately labeled antigen.

Lymphocytes can be obtained by removing the spleen or lymph nodes of sensitized animals in a sterile fashion and carrying out fusion. Alternately, lymphocytes can be stimulated or immunized i vitro, as described, for example, in C. Reading

J. Immunol. Meth. 53:261-291 1982.

A number of cell lines suitable for fusion have been developed, and the choice of any particular line for hybridization protocols is directed by any one of a number of criteria such as speed, uniformity of growth characteristics, deficiency of its metabolism for a component of the growth medium, and potential for good fusion frequency.

Intraspecies hybrids, particularly between like strains, work better than interspecies fusions. Several cell lines are available, including mutants selected for the loss of ability to secrete myeloma immunoglobulin. Included among these are the following mouse myeloma lines: MPC, ,-X45-6TG, 3-NSl-l-Ag4-l, P3-X63-Ag8, or mutants thereof such as

X63-Ag8.653, SP2-0-Agl4 (all BALB/C derived), Y3-'Agl.2.3

(rat) , and U266 (human) . Cell fusion can be induced either by virus, such as Epstein-Barr or Sendai virus, or polyethylene glycol. Polyethylene glycol (PEG) is the most efficacious agent for the fusion of mammalian somatic cells. PEG itself may be toxic for cells, and various concentrations should be tested for effects on viability before attempting fusion. The molecular weight range of PEG may be varied from 1,000 to

6,000. It gives best results when diluted to from about 20% to about 70% w/w in saline or serum-free medium. Exposure to

PEG at 37°C for about 30 seconds is preferred in the present case, utilizing murine cells. Extremes of temperature (i.e. above 45°C) are avoided, and preincubation of each component of the fusion system at 37°C prior to fusion gives optimum results. The ratio between lymphocytes and malignant cells is optimized to avoid cell fusion among spleen cells and a range of from about 1:1 to about 1:10 gives good results.

The successfully fused cells can be separated from the myeloma line by any technique known by the art. The most common and preferred method is to choose a malignant line which is Hypoxanthine Guanine Phosphoribosyl Transferase

(HGPRT) deficient, which will not grow in an aminopterin- containing medium used to allow only growth of hybrids and which is generally composed of hypoxanthine 1x10 -4M, aminopterin 1x10 5M, and thymidme 3x10-5M, commonly known as the HAT medium. The fusion mixture can be grown in the

HAT-containing culture medium immediately after the fusion

24 hours later. The feeding schedules usually entail maintenance in HAT medium for two weeks and then feeding with either regular culture medium or hypoxanthine, thymidine- containing medium.

The growing colonies are then tested for the presence of antibodies that recognize the antigenic preparation. Detection of hybridoma antibodies can be performed using an assay where the antigen is bound to a solid support and allowed to react to hybridoma supernatants containing putative antibodies. The presence of antibodies may be detected by "sandwich" techniques using a variety of indicators. Most of the common methods are sufficiently sensitive for use in the range of antibody concentrations secreted during hybrid growth.

Cloning of hybrids can be carried out after 21-23 days of cell growth in selected medium. Cloning can be performed by cell limiting dilution in fluid phase or by directly selecting single cells growing in semi-solid agarose. For limiting dilution, cell suspensions are diluted serially to yield a statistical probability of having only one cell per well. For 'the agarose technique, hybrids are seeded in a semisolid upper layer, over a lower layer containing feeder cells. The colonies from the upper layer may be picked up and eventually transferred to wells.

Antibody-secreting hybrids can be grown in various tissue culture flasks, yielding supernatants with variable concentrations of antibodies. In order to obtain higher concentrations, hybrids may be transferred into animals to obtain inflammatory ascites. Antibody-containing ascites can be harvested 8-12 days after intraperitoneal injection. The ascites contain a higher concentration of antibodies but include both monoclonals and immunoglobulins from the inflammatory ascites. Antibody purification may then be achieved by, for example, affinity chromatography.

The presence of the HIV-2/ST, its antigenic components or antibodies specific for same in a patient's serum can be detected utilizing antibodies prepared as above, either monoclonal or polyclonal, in virtually any type of immunoassay. A wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. This, of course, includes both single-site and two-site, or "sandwhich", assays of the non-competitive types, as well as in traditional competitive binding assays. Sandwich assays are among the most useful and commonly used assays and are favored for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention.

Briefly, in a typical forv/ard assay, an unlabeled antibody is immobilized in a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen binary complex, a second antibody, labeled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of a ternary complex of antibody-labeled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations on the forward assay include a simultaneous assay, in which both sample and labeled antibody are added simultaneously to the bound antibody, or a reverse assay in which the labeled antibody and sample to be tested are first combined, incubated and then added to the unlabeled surface bound antibody. These techniques are well known to those skilled in the art, and then possibly of minor variations will be readily apparent. As used herein, "sandwich assay" is intended to encompass all variations on the basic two-site technique. For example, these antibodies may be used to detect HIV-2/R0D, HIV-2/ST or HIV-2/ST-type viruses by use of inactivated forms of said viruses or their derivatives or with specific antigenic determinants or parts thereof as immobilized immunoadsorbants. Serum is obtained from subjects to be tested and said serum contacted to the immobilized viral immunoadsorbants. If said serum contains antibodies to said immunoadsorbants, an antibody-adsorbant conjugate will result. After removing excess serum and non-bound antibodies, .a second antibody specific to a first antibody, said first antibody being capable of forming a conjugate with said immunoadsorbant, is added thus resulting in a double antibody-adsorbant conjugate. This double antibody-adsorbant conjugate will only result if the test serum contains antibodies to the immunoadsorbant. Consequently, standard detection techniques can be used to identify the conjugate. The antigen may also be detected by a competitive binding assay in which a limiting amount of antibody specific for the molecule of interest (either an antigen or hapten) is combined with specified volumes of solutions containing an unknown amount of the molecule to be detected and a solution containing a detectably labeled known amount of the molecule to be detected or an analog thereof. Labeled and unlabeled molecules then compete for the available binding sites on the antibody. Phase separation of the free and antibody-bound molecules allows measurement of the amount of label present in each phase, thus indicating the amount of antigen or hapten in the sample being tested. A number of variations in this general competitive binding assay currently exist. -20-

Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten. The second antibody is linked to a reporter molecular which is used to indicate the binding of the second antibody to the hapten. By "reporter molecule", as used in the present specification and claims, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules. In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, B-galactosidase and alkaline phosphates, among others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. For example, p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates; for peroxidase conjugates, 1,2-phenylenediamine, 5-aminosalicyclic acid, or tolidine are commonly used. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labeled antibody is added to the first antibody hapten complex, allowed to bind, and then to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate -19- In any of the known immunoassays, for practical purposes, one of the antibodies or the antigen (virus or component thereof) will be typically bound to a solid phase and a second molecule, either the second antibody in a sandwich assay, or, in a competitive assay, the known amount of antigen, will bear a detectable label or reporter molecule in order to allow visual detection of an antibody-antigen reaction. When two antibodies are employed, as in the sandwich assay, it is only necessary that one of the antibodies be specific for HIV-2/ST or its antigenic components. The following description will relate to a discussion of a typical forward sandwich assay; however, the general techniques are to be understood as being applicable to any of the contemplated immunoassays.

In the typical forward sandwich assay, a first antibody having specificity for HIV-2/ST or its antigenic components is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs or microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing the molecule to the insoluble carrier. Following binding, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested in then added to the solid phase complex and incubated at 25°C for a period of time sufficient to allow binding of any subunit present in the antibody. The incubation period will vary but will generally be in the range of about 2-40 minutes. substrate is then added to the ternary complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present m the sample.

Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody absorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope. As in the EIA, the fluorescent labeled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining ternary complex is then exposed to the light of the appropriate wavelength, the fluorescence observed indicates the presence of the hapten of interest. Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed. It will be readily apparent to the skilled technician how to vary the procedure to suit the required purpose. It will also be apparent that the foregoing can be used to detect directly or indirectly (i.e. via antibodies) HIV-2/R0D, HIV-2, SIV and HIV-2/ST-type viruses.

The present invention also contemplates use of synthetic peptides to detect HIV-2/ST specific antibodies. The envelope protein is the most antigenic of viral proteins. It contains regions with characteristics consistent with being exposed on the viral surface and these regions are highly conserved. In accordance with the subject invention, a series of overlapping peptides from these regions can be synthesized and analyzed for their reactivity with sera from subjects to be tested. These peptides m an EIA assay can give a positive absorbance with seropositive serum at dilutions as high as 1:10,000. There are practical benefits of this approach. Because virus growth is not required, there is no risk to production personnel and the natural variability of different batches is removed. The ability to produce large quantities of peptide compared with virus, and the consistency of the chemistry, removes lot-to-lot variation. The vastly high molar peptide concentration achieved on the coated wells of the microplate results in the improved sensitivity, as well as the ability to accelerate the incubation timing so the assay can be performed in under one hour. It is therefore considered within the scope of the present invention to include any synthetic peptide or derivatives thereof prepared relative to any structural component of the HIV-2/ST virus particle wherein said structural component may be located on the surface or inside virus coat.

The subject invention also encompasses antibodies, either monoclonal or polyclonal, which are useful in the therapeutic control of infection by HIV-2/ST. Said antibodies may be prepared as described above and by injecting mammalian species, e.g. human, horse, rabbit, sheep, mice, etc. with inactivated virus, live virus, attenuated virus or parts or derivatives thereof and then purifying said antibodies employing the detection systems contemplated and described herein. Another aspect of the present invention is the genetic information contained in the RNA of HIV-2/ST. As defined herein, RNA, reverse transcriptase and DNA are referred to as the genetic components of the virus. Said RNA can be used to generate complimentary DNA and said DNA can be inserted in recombinant expression molecules such that certain genes encoded thereon are transcribed and the products can then be obtained. The HIV-2/ST DNA is also referred to as proviral DNA. Such products can then be used as antigenic components to generate, for example, antibodies.

Alternatively, said RNA and DNA can be used to generate probes to detect by hybridization cellular DNA from infected lymphocytes carrying integrated proviral DNA. In one embodiment, patient specimens containing whole lymphocytes are smeared onto a standard microscope slide, then fixed with an appropriate fixative. The DNA or RNA probe, which has been labeled (e.g. with biotin-avidin- enzyme) is added. The slide is then placed onto a heating block for one or two minutes to allow both the probe and the target nucleic acids to be separated from their complementary strand (if double stranded) . Non-hybridized probe DNA or RNA is removed by gentle washing. After a suitable detection complex is added, hybridization is detected with a light microscope following formation of a colored compound. Alternatively, the probe nucleic acid is labeled with a radioactive isotope and lymphocytes to be tested lyzed and their DNA fixed to, for example, nitrocellulose paper. Hybridization and DNA/RNA detection systems are well known in the art. In a further embodiment, the invention provides a cDNA comprising a region whose polynucleotide sequence is substantially:

AGTCGCTCTG CGGAGAGGCT GGCAGATTGA GCCCTGGGAG GTTCTCTCCA GCACTAGCAG GTAGAGCCTG GGTGTTCCCT GCTAGACTCT CACCAGTGCT TGGCCGGCAC TGGGCAGACG GCTCCACGCT TGCTTGCTTA AAAGACCTCT TAATAAAGCT GCCAGTTAGA AGCAAGTTAA GTGTGTGCTC CCATCTCTCC TAGTCGCCGC CTGGTCATTC GGTGTTCATC TAAAGTAACA AGACCCTGGT CTGTTAGGAC CCTTTCTGCT TTGGGAAACC AAGGCAGGAA AATCCCTAGC AGGTTGGCGC CCGAACAGGG ACTTGAAGAA GACTGAGAAG CCTTGGAACA CGGCTGAGTG AAGGCAGTAA GGGCGGCAGG AACAAACCAC GACGGAGTGC TCCTAGAAAA GCGCAGGCCG AGGTACCAAG GGCGGCGTGT GGAGCGGGAG TGAAAGAGGC CTCCGGGTGA AGGTAAGTGC CTACACCAAA TACAGTAGCC AGAAGGGCTT GTTATCCTAC CTTTAGACGG GTAGAAGATT GTGGGAGATG GGCGCGAGAA ACTCCGTCTT GAGAGGGAAA AAAGCAGACG AATTAGAAAA GATTAGGTTA CGGCCCGGCG GAAAGAAAAA ATATAGGCTA AAACATATTG TGTGGGCAGC GAATGAATTG GACAGATTCG GATTGGCAGA GAGCCTGTTG GAGTCAAAAG AGGGTTGCCA AAAAATTCTT ACAGTTTTAG ATCCATTAGT ACCGACAGGG TCAGAAAATT TAAAAAGCCT TTTTAATACT GTCTGCGTCA TTTGGTGTAT ACACGCAGAA GAGAAAGCGA AAGATACTGA AGAAGCAAAA CAAAAGGTAC AGAGACATCT AGTGGCAGAA ACAAAAACTA CAGAAAAAAT GCCAAGTACA AGTAGACCAA CAGCACCACC TAGCGGGAAC GGAGGAAACT TCCCCGTACA ACAAGTGGCC GGCAACTATA CCCATGTGCC ACTAAGTCCC CGAACCCTAA ATGCTTGGGT AAAACTAGTA GAGGAAAAGA AGTTCGGGGC AGAAGTAGTG CCAGGATTTC AGGCACTCTC AGAAGGCTGC ACGCCCTATG ATATTAATCA AATGCTTAAT TGTGTGGGCG ACCATCAAGC AGCTATGCAA ATAATCAGGG AAATTATTAA TGAAGAAGCA GCAGATTGGG ACGCACAACA CCCAATACCA GGCCCCTTAC CAGCGGGGCA GCTCAGGGAG CCAAGGGGAT CTGACATAGC AGGGACAACA AGCACAGTAG AAGAGCAGAT CCAGTGGATG TTTAGGCCAC AAAATCCTGT ACCAGTAGGA AGCATCTATA GAAGATGGAT CCAGATAGGG CTACAGAAGT GCGTCAGGAT GTACAACCCA ACCAACATCC TAGACATAAA ACAGGGACCA AAGGAGCCAT TCCAGAGTTA TGTAGATAGA TTCTACAAGA GCTTGAGGGC AGAACAAACA GATCCAGCAG TAAAAAATTG GATGACCCAA ACACTGCTAG TGCAGAATGC CAACCCAGAC TGTAAGTTAG TACTAAAAGG ACTAGGGATA AATCCTACCT TAGAAGAAAT GCTAACCGCC TGTCAGGGGG TAGGTGGACC AGGCCAGAAA GCCAGATTAA TGGCAGAAGC CTTAAAGGAG GCCATGGCAC CAGCCCCCAT CCCATTTGCA GCAGCCCAAC AGAGAAGGAC AATTAAGTGC TGGAATTGCG GAAAGGAAGG GCACTCGGCA AGACAATGCC GAGCACCTAG AAGACAAGGC TGCTGGAAAT GTGGCAAGGC AGGACACATC ATGGCAAAAT GCCCAGAAAG ACAGGCGGGT TTTTTAGGGT TGGGCCCATG GGGAAAGAAG CCCCGCAATT TCCCTGTGGC CCAAATCCCG CAGGGGCTGA CACCAACAGC ACCCCCGATA GACCCAGTAG AGGACCTACT AGAGAAGTAC ATGCAGCAAG GGAAAAGGCA GAGAGAGCAG AGAGAGAGGC CATACAAAGA AGTGACAGAG GACTTCCTGC AGGTCGAGAA ACAAGAGACA CCATGCAGAG AGACGACAGA GGACTTGCTG CACCTCAATT CTCTCTTTGG AAAAGACCAG TAGTCACAGC ACATGTTGAG GGCCAGCCAG TAGAAGTTTT GCTAGACACA GGGGCTGACG ACTCAATAGT AGCAGGCGTA GAGTTAGGGA GCAATTATAG TCCAAAGATA GTAGGGGGAA TAGGGGGATT CATAAATACC AAAGAATATA AAAATGTAGA AATAAGAGTA TTAAATAAAA GAGTAAGAGC CACCATAATG ACAGGTGATA CCCCAATCAA CATTTTTGGC AGAAACATTC TGACAGCCTT AGGCATGTCA TTAAATCTAC CAGTCGCCAA GATAGAACCA ATAAAAATAA TGCTGAAGCC AGGAAAGGAT GGACCAAAAC TGAGACAATG GCCCTTAACA AAAGAAAAAA TAGAGGCACT AAAAGAGATC TGTGAGAAAA TGGAAAGAGA GGGCCAGCTA GAGGAGGCAC CTCCAACTAA TCCTTATAAT ACCCCCACAT TTGCAATCAA GAAAAAGGAC AAAAACAAAT GGAGAATGCT AATAGATTTT AGAGAACTAA ACAAGGTAAC TCAAGACTTC ACAGAAATCC AGTTAGGAAT TCCACACCCA GCAGGACTAG CCAAGAAGAA ACGAATTACT GTCCTAGATG TAGGGGATGC TTACTTTTCC ATACCACTAC ATGAGGATTT TAGACAGTAT ACTGCATTTA CTCTACCATC AATAAACAAT GCTGAACCAG GAAAAAGATA CATATATAAA GTCTCACCAC AGGGATGGAA GGGATCACCA GCAATTTTTC AGTACACAAT GAGGCAGGTC TTAGAACCAT TCAGAAAAGC AAACCCGGAT ATCATTCTCA TTCAGTACAT GGATGATATC TTGATAGCCA GCGACAGGAC AGATTTAGAA CATGACAGAG TGGTTCTGCA GCTAAAGGAA CTTCTAAATG GCCTGGGATT TTCCACCCCA GATGAGAAGT TCCAAAAAGA CCCTCCATAC CAATGGATGG GCTATGAACT GTGGCCAACT AAATGGAAGC

TGCAAAGAAT ACAATTGCCC CAAAAGGAAG TATGGACAGT CAATGACATC

CAAAAACTGG TGGGTGTCCT AAATTGGGCA GCACAAATCT ACCCAGGGAT

AAAGACCAGA AACTTATGTA GGTTAATCAG AGGAAAAATG ACACTCACAG

AAGAGGTACA GTGGACAGAA TTAGCAGAAG CGGAACTAGA AGAAAACAAA

ATCATCTTAA GCCAGGAACA AGAAGGATGC TATTACCAAG AGGAAAAGGA

GCTAGAAGCA ACAGTCCAAA AAGATCAAGA CAATCAGTGG ACATATAAGA

TACACCAGGG AGGAAAAATT CTAAAAGTAG GAAAATATGC AAAGGTAAAA

AATACCCACA CCAACGGAGT CAGACTCCTA GCACAAGTAG TTCAAAAAAT

AGGAAAAGAA GCACTAGTCA TTTGGGGACG AATACCAAAA TTTCACCTAC

CAGTAGAAAG AGATACCTGG GAACAGTGGT GGGATAACTA CTGGCAAGTG

ACATGGATCC CAGACTGGGA CTTCATATCT ACCCCGCCAC TGGTCAGATT

AGTATTTAAC CTGGTGAAAG ATCCCATACT AGGCGCAGAA ACCTTCTACA

CAGATGGATC CTGCAATAAG CAATCAAGAG AAGGAAAAGC AGGATACATA

ACAGATAGAG GAAGAGACAA GGTGAGGCTA TTAGAGCAAA CCACCAATCA

GCAAGCAGAA TTAGAAGCCT TTGCGATGGC AGTAACAGAC TCAGGTCCAA

AGGCCAACAT TATAGTAGAC TCACAATATG TAATGGGAAT AGTAGCAGGC

CAACCAACAG AGTCAGAGAG TAAAATAGTA AATCAAATCA TAGAAGAAAT

GATAAAAAAG GAAGCAATCT ATGTTGCATG GGTCCCAGCC CATAAAGGCA

TAGGAGGAAA TCAGGAGGTA GATCACTTAG TAAGTCAGGG CATCAGACAA

GTATTATTCC TAGAGAAAAT AGAACCCGCT CAGGAGGAAC ATGAAAAATA

TCATAGCAAT GTAAAAGAAC TATCCCATAA ATTTGGACTG CCCAAATTAG

TGGCAAGACA AATAGTAAAC ACATGCACCC AATGTCAGCA GAAAGGGGAG

GCTATACATG GGCAAGTAAA TGCAGAATTA GGCACTTGGC AAATGGACTG

CACACACTTA GAAGGAAAAA TCATTATAGT AGCAGTACAT GTTGCAAGTG

GATTTATAGA AGCAGAAGTC ATCCCACAGG AATCAGGAAG GCAAACGGCA

CTCTTCCTAC TAAAACTGGC CAGTAGGTGG CCAATAACAC ATTTGCACAC

AGACAATGGT GCCAACTTCA CTTCACAGGA AGTAAAGATG GTGGCATGGT

GGATAGGTAT AGAACAATCC TTCGGAGTAC CTTACAATCC ACAAAGCCAA

GGAGTAGTGG AAGCAATGAA TCACCACCTA AAAAATCAGA TAAGCAGAAT

TAGAGAGCAG GCAAACACAG TAGAAACAAT AGTACTAATG GCAGTTCATT

GCATGAATTT TAAAAGGAGG GGAGGAATAG GGGATATGAC CCCAGCAGAA AGACTAATCA ATATGGTCAC TGCAGAACAG GAAATACAAT TCCTCCAAGC

AAAAAATTCA AAATTACAAA ATTTTCGGGT CTATTTCAGA GAAGGCAGAG

ATCAGCTGTG GAAAGGACCT GGGGAACTAC TGTGGAAGGG GGACGGAGCA

GTCATAGTCA AGGTAGGGGC TGACATAAAA ATAATACCAA GAAGGAAAGC

TAAGATCATC AAAGACTATG GAGGAAGGCA AGAGATGGAT AGCGGTTCCA

ACTTGGAGGG TGCCAGGGAG GATGGAGAGG TGGCATAGCC TTATCAAGTA TCTAAAATAC AGAACAGGAG ATCTAGAGAA GGTGTGCTAT GTTCCCCACC ATAAGGTGGG ATGGGCGTGG TGGACTTGCA GCAGGGTAAT ATTCCCATTA AAAGGAGAAA GTCATCTGGA GATACAGGCA TACTGGAACC TAACACCAGA AAAAGGATGG CTCTCCTCCT ATTCAGTAAG ACTAACTTGG TATACAGAAA AATTCTGGAC AGATGTTACC CCAGACTGTG CGGACTCCCT AATACATAGC ACTTATTTCT CTTGCTTTAC GGCAGGCGAA GTAAGAAGAG CCATCAGAGG GGAAAAGCTA TTATCCTGCT GCAACTACCC CCAAGCCCAT AAGTACCAGG TACCGTCACT CCAGTTTCTG GCCTTAGTGG TAGTGCAACA AAATGGCAGG CCCCAGAGAG ACAATACCAC CAGGAAACAG TGGCGAAGAA ACTATCGGAG AGGCCTTCGA GTGGCTAGAC AGGACGGTAG AAGCCATAAA CAGAGAGGCA GTGAACCACC TGCCCCGAGA GCTTATTTTC CAGGTGTGGC AAAGGTCCTG GAGATACTGG CATGATGAAC AAGGAATGTC AATAAGTTAC ACAAAGTATA GATATTTGTG CCTAATGCAG AAAGCTATGT TCATACATTC TAAGAGAGGG TGCACTTGCC TGGGGGGAGG ACATGGGCCG GGAGGATGGA GATCAGGACC TCCCCCTCCT CCCCCTCCAG GTCTAGTCTA ATGACTGAAG CACCAACAGA GTCTCCCCCG GAGGATAGGA CCCCACCGAG GGAGCCAGGG GATGAGTGGG TAATAGAAAC CCTGAGAGAG ATAAAATAAG AAGCTTTAAA GCACTTTGAC CCTCGCTTGC TAATTACTCT TGGCAACTAT ATCTATGCTA GACATGGAGA CACCCTTGAA GGCGCCAGAG GGCTCATTAG GATCCTACAA CGAGCCCTCC TCTTGCACTT CAGAGCAGGA TGCGGCCGCT CAAGGATTGG TCAGCCCAGG GGACGAAATC CTTTATCAGC TATACCAACC CCTAGAGGCA TGCGATAACA AATGTTACTG TAAAAAGTGC TGCTACCATT GCCAGATGTG TTTTTTAAAC AAGGGGCTCG GGATATGGTA TGAACGAAAG GGCAGAAGAA GAAGAACTCC GAAGAAAACT AAGGCTCATT CGTCTTCTGC ATCAGACAAG TGAGTAAGAT GTGTGGTAGG AATCAACTAT TTGTTGCCAG CTTGCTAGCT AGTGCTTGCT TAATATATTG CGTCCAATAT GTGACTGTTT TCTATGGCGT GCCCGTGTGG AGAAATGCAT CCATTCCCCT CTTTTGTGCA ACTAAAAATA GAGATACTTG

GGGAACCATA CAGTGCTTGC CAGACAATGA TGACTATCAG GAAATAGCTT

TAAATGTGAC AGAGGCCTTC GACGCATGGA ATAATACAGT AACAGAACAA

GCAGTAGAAG ATGTCTGGAG TCTATTTGAG ACATCAATAA AACCATGCGT

CAAACTAACA CCCTTATGTG TAGCAATGCG TTGTAACAGC ACAACTGCAA

AAAACACAAC CTCCACACCA ACAACCACCA CAACAGCAAA CACAACAATA

GGAGAGAATT CTTCATGCAT ACGCACAGAC AACTGCACAG GGTTGGGAGA

AGAAGAGATG GTCGACTGTC AGTTCAATAT GACAGGATTA GAGAGGGATA

AGAAAAAACT ATATAATGAA ACATGGTACT CAAAAGATGT AGTCTGTGAA

TCAAATGACA CCAAGAAAGA GAAAACATGT TACATGAACC ACTGCAACAC

ATCAGTCATC ACAGAGTCAT GTGACAAGCA CTATTGGGAT ACTATGAGGT

TTAGATATTG TGCACCACCG GGTTTTGCCC TGCTAAGATG CAATGATACC

AATTATTCAG GCTTTGAGCC CAATTGTTCT AAGGTAGTAG CTGCTACATG

TACAAGGATG ATGGAAACGC AAACCTCCAC TTGGTTTGGC TTTAATGGCA

CCAGGGCAGA AAATAGAACA TATATCTATT GGCATGGTAG GGATAATAGA

ACCATCATTA GCTTAAACAA GTTTTATAAT CTCACCGTAC ATTGTAAGAG

GCCAGGAAAC AAGACAGTTG TACCAATAAC ACTCATGTCA GGGTTAGTGT

TTCACTCCCA GCCAATCAAT AGAAGACCCA GGCAAGCATG GTGCTGGTTC

AAAGGCGAGT GGAAGGAAGC CATGAAGGAG GTGAAGCTAA CCCTTGCAAA

ACATCCCAGG TATAAAGGAA CCAACGACAC AGAAAAAATT CGTTTTATAG

CGCTAGGAGA ACGCTCAGAC CCAGAAGTGG CATACATGTG GACTAACTGC

AGAGGAGAAT TTCTCTACTG CAATATGACT TGGTTCCTCA ATTGGGTAGA

AAACAGAACG AATCAGACAC AGCACAATTA TGTGCCATGC CATATAAAGC

AAATAATTAA TACCTGGCAC AAGGTAGGGA AAAATGTATA TTTGCCTCCT

AGGGAAGGAC AGTTAACCTG CAACTCTACA GTGACCAGCA TAATTGCTAA

CATTGACGGA GGAGAGAACC AGACAAATAT TACCTTTAGT GCAGAGGTGG

CAGAACTATA CCGATTAGAA TTGGGGGATT ATAAATTGAT AGAAGTAACA

CCAATTGGCT TTGCACCTAC ACCAGTAAAA AGATACTCCT CTGCTCCAGT

GAGGAATAAA AGAGGTGTAT TCGTGCTAGG GTTCTTAGGT TTTCTCACGA

CAGCAGGAGC TGCAATGGGC GCGGCGTCCT TGACGCTGTC GGCTCAGTCT

CGGACTTTAT TGGCCGGGAT AGTGCAGCAA CAGCAACAGC TGTTGGACGT

GGTCAAGAGA CAACAAGAAA TGTTGCGACT GACCGTCTGG GGAACAAAAA ATCTCCAGGC AAGAGTCACT GCTATCGAGA AATACTTAAA GGACCAGGCG

CAACTAAATT CATGGGGATG TGCGTCTAGA CAAGTCTGCC ACACTACTGT

ACCATGGGTA AATGACACCT TAACGCCTGA TTGGAACAAC ATGACATGGC

AGGAATGGGA GCAACGAATC CGCAACCTAG AGGCAAATAT CAGTGAAAGT

TTAGAACAGG CACAAATCCA GCAAGAAAAG AACATGTATG AACTACAAAA

ATTAAATAGC TGGGATGTTT TTGGCAACTG GTTTGATTTA ACCTCCTGGA TCAAATATAT TCAGTATGGA GTTTATATAG TAGTAGGAAT AATAGTTTTA AGAATAGTAA TATATGTAGT ACAAATGTTA AGTAGACTTA GAAAGGGCTA TAGGCCTGTT TTCTCTTCCC CCCCCGCTTA CTTCCAACAG ATCCATATCC ACAAGGACCG GGAACAGCCA GCCAGAGAAG AAACAGAAGA AGACGTTGGA AACAGCGTTG GAGACAATTG GTGGCCCTGG CCGATAAGAT ATATACATTT CCTGATCCGC CAGCTGATTC GCCTCTTGAA CAGACTATAC AACATCTGCA GGGACTTACT ATCCAGGAGC TTCCAGACCC TCCAACTAAT CTCCCAGAGT CTTCGGAGAG CATTGACAGC AGTCAGAGAC TGGCTGAGAT TTAACACAGC CTACCTGCAA TATGGGGGCG AGTGGATCCA AGAAGCGTTC CGAGCCTTCG CGAGGGCTAC GGGAGAGACT CTTACAAACG CCTGGAGAGG CTTCTGGGGC ACACTGGGAC AAATTGGGAG GGGAATACTT GCAGTCCCAA GAAGGATCAG GCAGGGGGCA GAAATCGCCC TCCTGTGAGG GACGGCGGTA TCAACAGGGA GATTTTATGA ATACCCCATG GAGAGCCCCA GCAGAAGGGG AGAAAGGCTC GTACAAGCAA CAAAATATGG ATGATGTAGA TTCAGATGAT GATGACCTAG TAGGGGTCCC TGTCACACCA AGAGTACCAT TAAGAGAAAT GACATATAGG TTGGCAAGAG ATATGTCACA TTTGATAAAA GAAAAGGGGG GACTGGAAGG GCTGTATTAC AGTGATAGGA GACGTAGAGT CCTAGACATA TACTTAGAAA AGGAAGAGGG AATAATTGGA GACTGGCAGA ACTATACTCA TGGACCAGGA GTAAGGTATC CAAAGTTCTT TGGGTGGTTA TGGAAGCTAG TACCAGTAGA TGTCCCACAA GAGGGAGATG ACAGTGAGAC TCACTGCTTA GTGCATCCAG CACAAACAAG CAGGTTTGAT GACCCGCATG GAGAAACATT AGTTTGGAGG TTTGACCCCA CGCTAGCTTT TAGCTACGAG GCCTTTATTC GATACCCAGA GGAGTTTGGG TACAAGTCAG GCCTGCCAGA GGATGAATGG AAGGCAAGAC TGAAAGCAAG AGGGATACCG TTTAGCTAAA AACAGGAACA GCTATACTTG GTCAGGGCAG GAAGTAACTA ACAGAAAACA GCTGAGACTG CAGGGACTTT CCAGAAGGGG CTGTTACCAG GGGAGGGACA TGGGAGGAGC CGGTGGGGAA CGCCCTCATA CTTTCTGTAT AAATGTACCC GCTACTCGCA TTGTATTCAG

TCGCTCTGCG GAGAGGCTGG CAGATTGAGC CCTGGGAGGT TCTCTCCAGC ACTAGCAGGT AGAGCCTGGG TGTTCCCTGC TAGACTCTCA CCAGTGCTTG GCCGGCACTG GGCAGACGGC TCCACGCTTG CTTGCTTAAA AGACCTCTTA ATAAAGCTGC CAGTTAGAAG CA

In a further embodiment, the present invention also relates to a kit for the detection of HIV-2/ST and related viruses (HIV-2 family) , the kit being compartmentalized to receive a first container containing an inactivated virus or component thereof, a second container containing an antibody having specificity for said virus or component thereof, and a third container containing an antibody specific for first antibody and being labeled with a reporter molecule capable of giving a detectable signal. If the reporter molecule is an enzyme, then a fourth container containing a substrate for said enzyme is provided.

The HIV-2/ST virus has been deposited with the American Type Culture Collection (ATCC) and has been accorded the accession number

The bacteria carrying the recombinant clones comprising the full-length HIV-2/ST proviral DNA have similarly been deposited with the ATCC. JSP4-27 has been accorded the accession number , JSP4-32 has been accorded the accession number , and JS 4-34 has been accorded the accession number .

The subject invention is further described by the following examples.

EXAMPLE 1 ISOLATION OF HIV-2/ST

The approach outlined below uses standard biochemical techniques unless otherwise specified. Based on serological survey, peripheral blood was obtained from four healthy Senegalese prostitutes exhibiting seropositivity for the HIV-2/ROD family of viruses. Mononuclear cell preparations from the isolated blood samples were co-cultivated with normal human T-lymphocytes that had been stimulated with phytohemagglutinin (PHA) using the technique described by Barre-Sinoussi et al_. (Science

220:868, 1983) . T-cell growth factor (interleukin-2 (IL-2)) and goat anti-serum to -interferon were present in the medium. After one to two weeks, reverse transcriptase (RT) activity was detected in the culture supernatant from three of the four blood sources. Additionally, viral-specific mRNA was detected using in situ hybridization (See Example 2) .

The culture supernatant was subjected to polyethylene glycol

(PEG) precipitation and putative viral concentrates used to inoculate cultures of the same T-lymphocytes. This cycle of repeated PEG mediated virus precipitation which in turn were incubated repeatedly with the same immortalized T-lymphocyte cultures continued for from about one to about ten weeks.

After the last cycle, the viral concentrate from one (patient

ST) of the remaining three cultures was successfully used tc transmit the infection to four immortalized T-cell lines

SUPTT1, HUT78, H9 and CEMX174. In particular, Hut78, SupTl and CEMX174 were highly infected and immortalized with the virus. As with HIV-1, only T4(CD4) -enriched T-lymphocytes could produce the virus. On the basis of ultrastructural morphology, particulate RT activity, T4 cell tropism and antigenic reactivity by Western blot hybridization analysis, the virus was related to both HIV-2/ROD and SIV M..A_C„. The virus is henceforth designated HIV-2/ST. The immortalized T-cell lines infected with HIV-2/ST were designated Hut78/Bl2, SupTl/LKOOl and CEMX174/ST. A photograph of HIV-2/ST viral particles is shown in Figure 1.

EXAMPLE 2

ANALYSIS OF HIV-2/ST

The following example determines the relative nucleotide sequence homology of HIV-2/ST to HIV-2/ROD and

SIV(HTLV-IV) -type viruses. The latter type of viruses are those described by Kanki et _l. supra and previously thought to be an HTLV-IV virus.

Southern blot hybridization was at 37°C for 18 h with DNA probe (specific for HIV-2 and SIV(HTLV-IV) ) at 10 x

10 6 c.p.m. ml—1 (specific activity of probe was 2-3 x 108 c.p.m. ug~ DNA) in 2.4 x SSC, 40% (v/v) formamide and 10%

(w/v) dextran sulfate (See Shaw et a_l. , supra) . Filters were washed in 1 x SSC + 0.2% (w/v) sodium lauryl sulfate at 65°C. for 3 h and exposed to Kodak XAR-5 film for 4 h.

The Southern blot is shown in Fig. 2 and shows the relative nucleotide sequence homology of HIV-2/ST to

HIV-2/ROD (panel a) and SIV (HTLV-IV) (panel b) . Panels a and b are identical except for the viral-specific nucleic acid probe used, HIV-2/ROD in panel a and SIV(HTLV-IV) in panel b. HIV-2/ST was isolated as described in Example 1 followed by transmission to Hut78 cells (lanes 3-5) or SupTl cells (lane 6) . Total cellular and viral DNA was extracted from infected and uninfected cells as described by Shaw et al. supra. Approximately 10 ug of each DNA was digested with

Bam Hi, separated by 0.8% (w/v) agarose electrophoresis and transferred to two nitrocellulose filters. One filter (panel a) was hybridized to a 4.3 kbp HIV-2 specific probe from pROD35 (Guyader et al. supra) and the other filter (panel b) — to a corresponding 7-5 kbp SIV(HTLV-IV) specific probe from

PKE102 (Hahn et al. supra) .

The Southern blot (Fig. 2) of HIV-2/ST infected cellular DNA from the cultures described in Example 1 clearly show that HIV-2/ST is more related to HIV-2/R0D than to SIV(HTLV-IV) . The proviral DNA of HIV-2/ST hybridizes preferentially to HIV-2/ROD and the restriction pattern of this DNA is unique. Interestingly, two related yet distinguishable viral genotypes were found in HIV-2/ST, reflecting a genotypic heterogeneity characteristic of both HIV-1 and HIV-2. Importantly, SIV was not detected in any of four Senegal cultures of Example 1.

EXAMPLE 3

GENETIC ANALYSIS OF HIV-2/ST Figure 3 depicts further genetic characterization of HIV-2/ST by differential nucleic acid hybridization (Panel A) , restriction enzyme analysis of isolated DNA (Panel B) and comparative restriction enzyme mapping of full-length proviral DNA clones (Panel C) . Panel A: Identical nitrocellulose filters containing agarose gel separated restriction digests, 10 ug DNA each, of virus isolate HIV-1/IIIb (lanes 1) , HIV-2/ROD (lanes 2) , SIV _c-251 (see ref. 20) (lanes 3) , normal human lymphocyte DNA (lanes 4) , HIV-2/ST (cell line SupTl/LKOOl) (lanes 5) , and HIV-2/ST

(cell line Hut78/Bl2) (lanes 6) were prepared as described in Example 2. Hybridization was to 32P-labelled 4.3 kilobase

Pstl-Pstl (pol-central region-env) fragment of HIV-2/R0D probe and filters were washed at low (3 x SSC, 0.2% SDS,

55°C) or high (0.1 x SSC, 0.2% SDS, 65°C) stringency. Panel

B: Southern blot-hybridization restriction cleavage analysis using the same probe as for panel A and performed as described (21, 22) . SupTl/LKOOl is a permanent producing HIV-2/ST infected cell line derived by repeated PEG precipitation of primary HIV-2/ST coculture supernatants onto SupTl cells. Hut78/Bl2 is an HIV-2/ST producing cell line derived by single cell cloning of HIV-2/ST infected Hut78 cells, also originally obtained by repeated PEG precipitations of primary lymphocyte co-culture supernatants. Restriction enzymes were Xbal (lanes 1) , Xhol (lanes 2) , BamHI (lanes 3) , EcoRI (lanes 4) , Hindi (lanes 5) , and Kpn I (lanes 6) . Panel C: Restriction maps of three full-length recombinant proviral DNA clones of HIV-2/ST (JSP4-27; JSP4-32; JSP4-34) obtained by Sau3AI (Mbol) partial digestion of Hut78/Bl2 DNA and ligation into Jl-lambda phase, as described (Example 8) . Restriction enzymes shown are Pstl (P) , BamHI (B) , Xhol (0) , Bglll (G) , EcoRI (E) , Hindlll (H) , Xbal (X) , Sail (L) , and Sstl (S) . Asterisks denote sites in HIV-2/R0D or SIV^ that are also present in the HIV-2/ST clones. Dotted lines denote flanking cellular sequences in the HIV-2/ST clones.

The results show that the DNA genome of the HIV-2/ST virus hybridized at low and high stringency to the prototype HIV-2/R0D clone whereas SIV hybridized to the same HIV-2/R0D probe only under conditions of low stringency. HIV-1 did not hybridize to the HIV-2/R0D probe under any conditions. Conversely, clones of SIV used as probe hybridized to HIV-2/R0D and HIV-2/ST with equal intensity but only under conditions of low stringency. A full-length HTLV-1 probe gave no hybridization. Two different HIV-2/ST producing cell lines derived from the transmission of cell-free virus from primary ST lymphocyte co-cultures exhibited very similar proviral restriction enzyme cleavage patterns but were distinguishable by polymorphic Xbal and

BamHI sites. Comparative restriction mapping of the three full-length HIV-2/ST proviral clones confirmed the genetic similarity between HIV-2/ST and HIV-2/R0D (10 of 25 restriction sites in common) and the dissimilarity between

HIV-2/ST and SIV (2 of 36 restriction sites in common) .

Furthermore, one of these proviral DNA clones (lambda

JSP-27) , when transfected into T-lymphocyte cell lines, produced virus that was replication-competent and infectious and whose biological properties were identical to the parental virus isolate. Together, these studies showed conclusively that the HIV-2/ST virus is genotypically more related to prototype HIV-2/ROD than to other primate viruses, that within the HIV-2/ST virus isolate there exist polymorphic replication competent viral genotypes, and trhat an SIV-like virus is not present in the HIV-2/ST cultures.

EXAMPLE 4

WESTERN BLOT AND RADIOIMMUNOPRECIPITATION ANALYSES

The structural, antigenic and functional characteristics of HIV-2/ST proteins were evaluated by Western blot and radioimmunoprecipitation (RIP) analyses and the results are shown in Figure 4.

Western immunoblots were performed as described (Bernsteins et al. J. Gen. Virol. 67:1601, 1986) using a human (West African) anti-HIV-2 serum (lanes 1-8) and a human anti-HIV-1 serum (lanes 9-12) . Bound antibody was detected with peroxidase-conjugated goat anti-human immunoglobulin. Antigen preparations were viral lysates from HIV-2/ST line SupTl/LKOOl (lanes 1, 12) ; single cell derived clones from SupTl/LKOOl designated ST.17 (lane 2) , ST.9 (lane 3) , ST.24 (lanes 4, 11) ; HIV-2/ROD (lanes 5, 10) ; SIV_]yiAC (lanes 6) ; HIV-1/IIIb (lanes 7, 9) ; uninfected control cells (lane 8) . Panel B: Immunoprecipitation of HIV-2/ST envelope proteins from cloned cell lines of infected Sup-Tl cells shown in

Panel A. Cell lysates from 35S-cysteine and 35S-methionine labeled clones were prepared and immunoprecipitated as described previously (Hoxie et al. Science 234:1123, 1986) , using either normal human serum (lanes 1, 3, 5, 7), or serum from a West African patient with HIV-2 infection (lanes 2, 4,

6, 8). Shown are clones designated ST.9 (lanes 1 and 12), ST.17 (lanes 3 and 4) _r ST.23 (lanes 5 and 6) which exhibit an envelope precursor molecule of approximately 180 kD and ST.24

(lanes 7 and 8) which exhibit an envelope precursor molecule of 170 kD. The smaller sized envelope precursor (gpl70) of ST.24 corresponds to the truncated transmembrane protein

(gp30) of the same virus (Panel A) . Panel C:

Co-precipitation of HIV-2/ST envelope glycoproteins with CD4. 35 35

Cell lysates from S-cysteme and S-methionine-labeled uninfected SupTl cells (lanes 1, 2, 3, 4, 5) and uncloned

SupTl/LKOOl cells (lanes 6, 7, 8, 9, 10) were prepared and immunoprecipitated as described (25) , using the nonreactive monoclonal antibody OKT3 (lanes 1 and 6) , OKT4 (lanes 2 and

7) , OKT4A (lanes 3 and 8) , normal human serum (lanes 4 and

9) , or human serum from a patient with HIV-2 infection (lanes

5 and 10) . Both OKT4 and OKT4A immunoprecipitate the 56 kD

CD4 molecule from the uninfected cells. However, in the infected cells OKT4 (but not OKT4A) immunoprecipitates CD4 as well as three high molecular weight molecules corresponding to viral envelope gene products that are also immunoprecipitated by immune human serum (lane 10) .

EXAMPLE 5

SYNCYTIUM FORMATION BY HIV-2/ST Figure 5 shows syncytium formation by HIV-2/ST, HIV-2/ROD, and HIV-1/IIIb infected cells and CD4 positive SupTl (Panel A) and Hela-T4 (Panel B) indicator cells. Panel

A: Virally-infected SupTl or H9 cells were mixed 1:5 with uninfected SupTl cells, incubated 24 hours, and photographed.

Results were identical at 48 hours. Panel B: Same as Panel

A except that virally-infected cells and Hela-T4 cells were mi.xed 1:2, allowed to adhere to plastic for 5 hours, non-adherent cells washed away, and the cultures evaluated at

24 hours and again at 48 hours. Both assays were performed as described (Maddon et al. Cell 47:333, 1986; Lifson et al.

Science 232:1123, 1986) . Leu3a/OKT4a antibodies completely blocked syncytium formation with HIV-1/IIIb and HIV-2/ROD whereas Leu2a/0KT8 had no effect, and Hela cells lacking CD4 gave no syncytia. The parental HIV-2/ST cell line (ST/LK001) and single cell derived clones (ST.9 and ST.24) are described in Example 3.

EXAMPLE 6

CYTOPATHIC/CELL KILLING PROPERTIES OF HIV-2/ST

Figure 6 shows a comparison of the cytopathic/cell killing properties of HIV-2/ST and prototype HIV-1 (isolates

Illb, BC, WMJ, RH) and HIV-2 (isolate ROD) viruses. Each panel summarizes a 21 day experiment in which equivalent amounts of cell-free concentrated virus (250,000 c.p.m. RT activity) were applied to cultures of uninfected SupTl cells

(Panels A, B) or PHA-stimulated peripheral blood lymphocytes

(Panels C, D) . In Panel E, 7,500 c.p.m. RT activity of all viruses were used except for HIV-2/ST 10 x (75,000 c.p.m.) and HIV-2/ST 100 x (750,000 c.p.m.) . Supernatant reverse transcriptase activity and percentage of cells infected with virus as determined by indirect immunofluorescence is shown over time. Cytopathicity (cell killing) was determined by counting viable cell numbers using hemocytometer with trypan blue exclusion and automated cell counter. All cell counts are adjusted to reflect culture splits done weekly to keep total cell concentration of control cells between 0.5 x 10 and 1.0 x 10 /ml. Syncytium formation was scored from 0

(none) to 4+ (extensive) and is shown in brackets. Mock infected culture supernatants lacking virus served as controls. Ten percent (w/v) final concentration of polyethylene glycol (PEG) was used in experiments shown in

Panels B, D, and E in order to precipitate virus and enhance virus-cell interaction. Comparable levels of virus infectivity were achieved with all viruses by day 14, but cytopathic effect as judged by viable cell counts and syncytium induction was much less for HIV-2/ST than for all other HIV-2 and HIV-1 viruses.

EXAMPLE 7

IN SITU HYBRIDIZATION ANALYSIS Figure 7 depicts an in situ hybridization analysis of cell-free viral infection by HIV-1/IIIb, day 1 post-infection (Panel A) ; HIV-2/R0D, day 1 post-infection (Panel B) ; HIV-2/ST, day 1 post-infection (Panel C) ; HIV-2/ST, day 10 post-infection (Panel D) . Identical amounts of virus (100,000 c.p.m. RT activity) were concentrated by PEG precipitation of culture supernatants, applied to 5 x 10 CEM x 174 cells, and cultured for 14 days. In situ hybridizations were performed as described (Gendelman e_t al. P.N.A.S. USA 82:7086, 1983) using polymerase-central region-envelope DNA probes from HIV-1 (BH-10 clone) ,

HIV-2/R0D, and HIV-2/ST nick-translated to the same specific activity of 2 x 10 8 d.p.m./ug (35S) . Hybridization was performed for 2 days and exposure to photographic emulsion for 3 days. Controls included HIV-1 and HIV-2 probes on uninfected cells and HTLV-1 probe on HIV-1 and HIV-2 infected cells. Data from this experiment are summarized in Table 1. 50,000 cells per slide were examined for viral mRNA production. Relative amounts of viral mRNA production per cell were scored qualitatively from absent silver grains (—) to grans too numerous to count (++++) . A marked delay in the initial development and spread of productive viral infection by HIV-2/ST is seen. However, those few HIV-2/ST cells that were productively infected at early time points expressed equal amounts of viral RNA on a per cell basis as compared with prototype HIV-1 and HIV-2 viruses.

TABLE 1

Days Post- Virus % of Cells Relative Amount Infection Isolate Expressing of Viral RNA per Viral RNA Infected Cell

1 HIV-1/IIIb 3% ++++

HIV-2/ROD 8% ++++

HIV-2/ST 0.01% ++++

Control 0% —

3 HIV-1/IIIb 35% ++++

HIV-2/ROD 45% ++++

HIV-2/ST 0.01% ++++

Control 0% —

7 HIV-1/IIIb 100% ++++

HIV-2/ROD 100% ++++

HIV-2/ST 0.5% ++++

Control 0% --

10 HIV-1/IIIb 100% ++++

HIV-1/ROD 100% ++++

HIV-2/ST 10% *+•+++

Control 0% __ 14 HIV-1 /Illb ND ND

HIV- 2 /ROD ND ND

HIV-2 /ST 50% ++++

Control 0 %

Table 1: In situ hybridization time course analysis of viral infection by HIV-1/IIIb, HIV-2/R0D, and HIV-2/ST.

EXAMPLE 8 CLONING HIV-2/ST PROVIRAL DNA

In accordance with the subject invention, three full-length HIV-2/ST proviral clones were obtained from an immortalized T-lymphocyte line infected with the virus. The infected cell line is designated Hut78/B12 and the clones obtained are designated JSP4-27, JSP4-32 and JSP4-34. Total cellular DNA from Hut78/B12 was isolated using standard techniques, subjected to Sau3A partial digestion and cloned into BamHl-digested Jl-lambda phase. A cosmid library as thus obtained in Escherichia coli. Clones carrying HIV-2/ST proviral DNA were identified by plaque hybridization using a full-length SIV probe (see Hahn e_t al. supra) . The three aforementioned clones were thus identified. Techniques useful in cloning the HIV-2/ST proviral DNA can be found in Maniatis et a_l. supra and in Shaw et al. supra. These clones provide a source of recombinant viral components when ligated into suitable expression vectors.

EXAMPLE 9 HTLV-IV/ST INFECTION OF T-LYMPHOCYTES

Fig. 8 is a graphical representation of the level of reverse transcriptase (RT) detected following days after infection of T-lymphocyte lines with HIV-2/ST. RT was measured using standard techniques in the supernatant fluid of the culture media. The graph clearly demonstrates the presence of HIV-2/ST in the immortalized T-lymphocyte lines SupTl and Hut78.

EXAMPLE 10 POLYCLONAL ANTIBODY PREPARATION

Rabbits are immunized by intradermal injection with

50 ul of Freund's complete adjuvant containing 20 ug/ml of

HIV-2/ST or its components in 10 locations along the back. The rabbits are first shaved on both sides of the back for easy intradermal injection. The antigen-adjuvant mixture is prepared by mixing in two connected 1 ml glass typhlon syringes and administered in 100 ul doses per location.

Forty days after injection rabbits are boosted by direct intravenous injection of 10 ug/100 ul PBS of antigen. Seven to. ten days later, rabbits are bled via the ear vein and sera tested for presence of anti-HIV-2/ST antibodies. Screening and titration of rabbit antisera is accomplished using various 125I-labeled components of HIV-2/ST in the presence of goat anti-rabbit coated latex beads.

EXAMPLE 11

MONOCLONAL ANTIBODY PRODUCTION

Monoclonal antibodies ar prepared in accordance with the techniques developed by Kohler and Mulskin (Eur. J.

Immunol. 6:511-519, 1976) . Mice are immunized with

HIV-2/ST or its components intraperitoneally with 10 ug of subunit in 100 ul of Freund's complete adjuvant. Two weeks after the initial injection, the mice are boosted with 10 ug of antigen in 100 ul of alum (10 mg/ml) by intraperitoneal injection of 10 ug of antigen in phosphate buffered saline

(PBS) . Five days after the last injection and after confirmation of the presence of antibody in mouse sera, the mice are sacrificed and their spleens removed. Spleen cells are obtained by gentle disruption of the spleen in a 7 ml

Dounce homogenizer in 3.5-4 ml PBS. The cells are then pelleted at 1200 rpm in a PR6 centrifuge for 6 minutes at room temperature. The supernatant is removed into a suction flask, and the cells are resuspended in 15 ml 0.83% NH^Cl. This suspension is incubated at room temperature for 5 minutes then underlain with 10 ml fetal calf serum at 37°C.

The cells are again pelleted by centrifugation for 8 minutes, at 1200 rpm at room temperature, then the supernatant is withdrawn into a suction flask and cells resuspended in 20 ml

PBS.

EXAMPLE 12

SANDWICH ASSAY FOR HIV-2/ST VIRUS PARTICLE

For detection of the presence of HIV-2/ST in serum, approximately 100 ul of a monoclonal antibody prepared as in

Example 3 is immobilized on latex beads and is contacted with about 100 ul of the serum sample to be tested. The antibody and serum are allowed to react for a period of about ten minutes and then rinsed with a solution of PBS. To the latex beads is then added about 100 ul of HIV-2/ST specific antibody conjugated to horseradish peroxidase. The labeled antibody bead mixture is incubated for a period of about ten minutes. At this time, an enzyme substrate, hydrogen peroxide and aminoantipyrine, are contacted with the beads, and this mixture is incubated for a period of about 5-10 minutes, at which time the development of color in the sample is an indication of a positive reaction and the presence of the HIV-2/ST. It is also possible to immobilize inactivated HIV-2/ST or component thereof to solid support (e.g. microliter well or latex bead) to form an immobilized immunoadsorbant. Said immunoadsorbant is then contacted with serum to be tested, said serum may contain antibodies to

HIV-2/ST or related viruses. After washing away the serum, antibody-immunoadsorbant complexes are detected by adding a second antibody specific for first antibody wherein said second antibody is labeled.

Claims

WHAT IS CLAIMED IS:

1. A novel virus isolated from serum or mononuclear cells from human subjects seropositive for the HIV-2/ROD virus, said virus having the characteristics of a retrovirus and being antigenically more similar to and genetically distinct from, HIV-2/R0D and SIV than to HIV-1.

2. The virus according to Claim 1 having the identifying characteristics of HIV-2/ST, accorded the and ATCC accession number .

3. The structural components of HIV-2/ST.

4. The structural components according to Claim 3 which comprise the extracellular envelope protein, outer core protein, major structural core protein, transmembrane envelope protein and lipid membrane.

5. The RNA and DNA encoding the structural components according to Claim 3 or 4.

6. The genetic components of HIV-2/ST.

7. The genetic components according to Claim 6 which comprises RNA, reverse transcriptase and DNA.

8. Recombinant DNA molecules comprising the full-length HIV-2/ST proviral DNA.

9. The recombinant molecules according to Claim 8 having the characteristics of JSP4-27, accorded the ATCC accession number _, JSP4-32, accorded the ATCC accession number , or JSP4-34, accorded the ATCC accession number

10. The recombinant viral components encoded by the DNA according to Claim 8 or 9.

11. Antibodies to the structural and genetic components of HIV-2/ST.

12. The antibodies according to Claim 11 wherein said antibodies are monoclonal or polyclonal.

13. The antibodies according to Claim 11 or 12 which are specific for extracellular envelope protein, outer core protein, major structural core protein, transmembrane envelope protein and lipid membrane.

14. Antibodies directed against the antibodies according to Claim 11.

15. Antibodies directed against the antibodies according to Claim 12.

16. Antibodies directed against the antibodies according to Claim 13.

17. The antibodies according to Claim 14 or 15 or 16 wherein said antibodies are monoclonal or polyclonal.

18. Immortalized T-lymphotcyte lines infected with the virus of Claim 1 or 2.

19. The immortalized T-lymphocyte line according to Claim 18 having the characteristics of Hut78/Bl2.

20. The immortalized T-lymphocyte line according to Claim 18 having the characteristics of SupTl/LKOOl.

21. The virus prepared from the immortalized T-lymphocyte lines according to Claim 18.

22. The virus prepared from the immortalized T-lymphocyle lines according to Claim 19 or 20.

23. A method for isolating a virus, said virus being antigenically more similar to, and genetically distinct from, HIV-2/R0D and SIV_MAC than to HIV-1, comprising the steps :

(a) isolating virus infected mononuclear cells from peripheral blood;

(b) contacting said mononuclear cells with non-infected lymphocytes in a culture medium; (c) isolating supernatant from said culture medium and concentrating virus;

(d) re-infecting the same lymphocyte in step (a) with the concentrated virus preparation of step (c) ;

(e) repeating steps (a) to (d) for a time sufficient to develop highly infected and immortalized lymphocyte line.

24. The method according to Claim 23 wherein said virus is HIV-2/ST.

25. The method according to Claim 23 wherein concentrating the virus in step (c) is by polyethylene glycol mediated virus precipitation.

26. The method according to Claim 23 wherein the non-infected lymphocytes in step (b) are selected from the group consisting of Hut78 , SupTl, CEMX174 and H9.

27. An infected and immortalized T-lymphocyte line prepared according to the method of Claim 23.

28. The T-lymphocyte line according to Claim 27 having the characteristics of Hut78/Bl2.

29. The T-lymphocyte line according to Claim 27 having the characteristics of SupTl/LKOOl.

30. A method of detection of HIV-2/ST and antigenically related viruses comprising the steps of contacting serum of individual to be tested with immobilized immunoadsorbant comprising HIV-2/ST or component thereof and detecting any resultant antibody-immunoadsorbant complex.

31. A method of detection of HIV-2/ST and antigenically related virus DNA in infected cells comprising the steps of lyzing said cells and fixing DNA onto solid support then detecting viral DNA by specific probe nucleic acid.

32. The method according to Claim 31 wherein said probe nucleic acid is RNA or DNA.

33. A kit for the diagnosis and monitoring of HIV-2/ST or HIV-2-type virus antibody, the kit being compartmentalized to receive: a first container containing an inactivated virus or component thereof, a second container containing a antibody having specificity for said virus or component thereof and a third container containing an antibody specific for first antibody and being labeled with a reporter molecule capable of giving a detectable signal.

34. The kit of Claim 33 wherein the reporter molecule is a radioisotope, an enzyme, a fluorescent molecule, a chemiluminescent molecule or a bioluminescent molecule.

35. The kit of Claim 33 wherein the reporter molecule is an enzyme.

36. The kit of Claim 35 wherein the kit further comprises a fourth container containing a substrate for the enzyme.

37. A cDNA comprising a region whose polynucleotide sequence is substantially AGTCGCTCTG CGGAGAGGCT GGCAGATTGA GCCCTGGGAG GTTCTCTCCA GCACTAGCAG GTAGAGCCTG GGTGTTCCCT GCTAGACTCT CACCAGTGCT TGGCCGGCAC TGGGCAGACG GCTCCACGCT TGCTTGCTTA AAAGACCTCT TAATAAAGCT GCCAGTTAGA AGCAAGTTAA GTGTGTGCTC CCATCTCTCC TAGTCGCCGC CTGGTCATTC GGTGTTCATC TAAAGTAACA AGACCCTGGT CTGTTAGGAC CCTTTCTGCT TTGGGAAACC AAGGCAGGAA AATCCCTAGC AGGTTGGCGC CCGAACAGGG ACTTGAAGAA GACTGAGAAG CCTTGGAACA CGGCTGAGTG AAGGCAGTAA GGGCGGCAGG AACAAACCAC GACGGAGTGC TCCTAGAAAA GCGCAGGCCG AGGTACCAAG GGCGGCGTGT GGAGCGGGAG TGAAAGAGGC CTCCGGGTGA AGGTAAGTGC CTACACCAAA TACAGTAGCC AGAAGGGCTT GTTATCCTAC CTTTAGACGG GTAGAAGATT GTGGGAGATG GGCGCGAGAA ACTCCGTCTT GAGAGGGAAA AAAGCAGACG AATTAGAAAA GATTAGGTTA CGGCCCGGCG GAAAGAAAAA ATATAGGCTA AAACATATTG TGTGGGCAGC GAATGAATTG GACAGATTCG GATTGGCAGA GAGCCTGTTG GAGTCAAAAG AGGGTTGCCA AAAAATTCTT ACAGTTTTAG ATCCATTAGT ACCGACAGGG TCAGAAAATT TAAAAAGCCT TTTTAATACT GTCTGCGTCA TTTGGTGTAT ACACGCAGAA GAGAAAGCGA AAGATACTGA AGAAGCAAAA CAAAAGGTAC AGAGACATCT AGTGGCAGAA ACAAAAACTA CAGAAAAAAT GCCAAGTACA AGTAGACCAA CAGCACCACC TAGCGGGAAC GGAGGAAACT TCCCCGTACA ACAAGTGGCC GGCAACTATA CCCATGTGCC ACTAAGTCCC CGAACCCTAA ATGCTTGGGT AAAACTAGTA GAGGAAAAGA AGTTCGGGGC AGAAGTAGTG CCAGGATTTC AGGCACTCTC AGAAGGCTGC ACGCCCTATG ATATTAATCA AATGCTTAAT TGTGTGGGCG ACCATCAAGC AGCTATGCAA ATAATCAGGG AAATTATTAA TGAAGAAGCA GCAGATTGGG ACGCACAACA CCCAATACCA GGCCCCTTAC CAGCGGGGCA GCTCAGGGAG CCAAGGGGAT CTGACATAGC AGGGACAACA AGCACAGTAG AAGAGCAGAT CCAGTGGATG TTTAGGCCAC AAAATCCTGT ACCAGTAGGA AGCATCTATA GAAGATGGAT CCAGATAGGG CTACAGAAGT GCGTCAGGAT GTACAACCCA ACCAACATCC TAGACATAAA ACAGGGACCA AAGGAGCCAT TCCAGAGTTA TGTAGATAGA TTCTACAAGA GCTTGAGGGC AGAACAAACA GATCCAGCAG TAAAAAATTG GATGACCCAA ACACTGCTAG TGCAGAATGC CAACCCAGAC TGTAAGTTAG TACTAAAAGG ACTAGGGATA AATCCTACCT TAGAAGAAAT GCTAACCGCC TGTCAGGGGG TAGGTGGACC AGGCCAGAAA GCCAGATTAA TGGCAGAAGC CTTAAAGGAG GCCATGGCAC CAGCCCCCAT CCCATTTGCA GCAGCCCAAC AGAGAAGGAC AATTAAGTGC TGGAATTGCG GAAAGGAAGG GCACTCGGCA AGACAATGCC GAGCACCTAG AAGACAAGGC TGCTGGAAAT GTGGCAAGGC AGGACACATC ATGGCAAAAT GCCCAGAAAG ACAGGCGGGT TTTTTAGGGT TGGGCCCATG GGGAAAGAAG CCCCGCAATT TCCCTGTGGC CCAAATCCCG CAGGGGCTGA CACCAACAGC ACCCCCGATA GACCCAGTAG AGGACCTACT AGAGAAGTAC ATGCAGCAAG GGAAAAGGCA GAGAGAGCAG AGAGAGAGGC CATACAAAGA AGTGACAGAG GACTTCCTGC AGGTCGAGAA ACAAGAGACA CCATGCAGAG AGACGACAGA GGACTTGCTG CACCTCAATT CTCTCTTTGG AAAAGACCAG TAGTCACAGC ACATGTTGAG GGCCAGCCAG TAGAAGTTTT GCTAGACACA GGGGCTGACG ACTCAATAGT AGCAGGCGTA GAGTTAGGGA GCAATTATAG TCCAAAGATA GTAGGGGGAA TAGGGGGATT CATAAATACC AAAGAATATA AAAATGTAGA AATAAGAGTA TTAAATAAAA GAGTAAGAGC CACCATAATG ACAGGTGATA CCCCAATCAA CATTTTTGGC AGAAACATTC TGACAGCCTT AGGCATGTCA TTAAATCTAC CAGTCGCCAA GATAGAACCA

ATAAAAATAA TGCTGAAGCC AGGAAAGGAT GGACCAAAAC TGAGACAATG GCCCTTAACA AAAGAAAAAA TAGAGGCACT AAAAGAGATC TGTGAGAAAA TGGAAAGAGA GGGCCAGCTA GAGGAGGCAC CTCCAACTAA TCCTTATAAT ACCCCCACAT TTGCAATCAA GAAAAAGGAC AAAAACAAAT GGAGAATGCT AATAGATTTT AGAGAACTAA ACAAGGTAAC TCAAGACTTC ACAGAAATCC AGTTAGGAAT TCCACACCCA GCAGGACTAG CCAAGAAGAA ACGAATTACT GTCCTAGATG TAGGGGATGC TTACTTTTCC ATACCACTAC ATGAGGATTT TAGACAGTAT ACTGCATTTA CTCTACCATC AATAAACAAT GCTGAACCAG GAAAAAGATA CATATATAAA GTCTCACCAC AGGGATGGAA GGGATCACCA GCAATTTTTC AGTACACAAT GAGGCAGGTC TTAGAACCAT TCAGAAAAGC AAACCCGGAT ATCATTCTCA TTCAGTACAT GGATGATATC TTGATAGCCA GCGACAGGAC AGATTTAGAA CATGACAGAG TGGTTCTGCA GCTAAAGGAA CTTCTAAATG GCCTGGGATT TTCCACCCCA GATGAGAAGT TCCAAAAAGA CCCTCCATAC CAATGGATGG GCTATGAACT GTGGCCAACT AAATGGAAGC TGCAAAGAAT ACAATTGCCC CAAAAGGAAG TATGGACAGT CAATGACATC CAAAAACTGG TGGGTGTCCT AAATTGGGCA GCACAAATCT ACCCAGGGAT AAAGACCAGA AACTTATGTA GGTTAATCAG AGGAAAAATG ACACTCACAG AAGAGGTACA GTGGACAGAA TTAGCAGAAG CGGAACTAGA AGAAAACAAA ATCATCTTAA GCCAGGAACA AGAAGGATGC TATTACCAAG AGGAAAAGGA GCTAGAAGCA ACAGTCCAAA AAGATCAAGA CAATCAGTGG ACATATAAGA TACACCAGGG AGGAAAAATT CTAAAAGTAG GAAAATATGC AAAGGTAAAA AATACCCACA CCAACGGAGT CAGACTCCTA GCACAAGTAG TTCAAAAAAT AGGAAAAGAA GCACTAGTCA TTTGGGGACG AATACCAAAA TTTCACCTAC CAGTAGAAAG AGATACCTGG GAACAGTGGT GGGATAACTA CTGGCAAGTG ACATGGATCC CAGACTGGGA CTTCATATCT ACCCCGCCAC TGGTCAGATT AGTATTTAAC CTGGTGAAAG ATCCCATACT AGGCGCAGAA ACCTTCTACA CAGATGGATC CTGCAATAAG CAATCAAGAG AAGGAAAAGC AGGATACATA ACAGATAGAG GAAGAGACAA GGTGAGGCTA TTAGAGCAAA CCACCAATCA GCAAGCAGAA TTAGAAGCCT TTGCGATGGC AGTAACAGAC TCAGGTCCAA AGGCCAACAT TATAGTAGAC TCACAATATG TAATGGGAAT AGTAGCAGGC CAACCAACAG AGTCAGAGAG TAAAATAGTA AATCAAATCA TAGAAGAAAT

GATAAAAAAG GAAGCAATCT ATGTTGCATG GGTCCCAGCC CATAAAGGCA

TAGGAGGAAA TCAGGAGGTA GATCACTTAG TAAGTCAGGG CATCAGACAA

GTATTATTCC TAGAGAAAAT AGAACCCGCT CAGGAGGAAC ATGAAAAATA

TCATAGCAAT GTAAAAGAAC TATCCCATAA ATTTGGACTG CCCAAATTAG

TGGCAAGACA AATAGTAAAC ACATGCACCC AATGTCAGCA GAAAGGGGAG

GCTATACATG GGCAAGTAAA TGCAGAATTA GGCACTTGGC AAATGGACTG

CACACACTTA GAAGGAAAAA TCATTATAGT AGCAGTACAT GTTGCAAGTG

GATTTATAGA AGCAGAAGTC ATCCCACAGG AATCAGGAAG GCAAACGGCA

CTCTTCCTAC TAAAACTGGC CAGTAGGTGG CCAATAACAC ATTTGCACAC

AGACAATGGT GCCAACTTCA CTTCACAGGA AGTAAAGATG GTGGCATGGT

GGATAGGTAT AGAACAATCC TTCGGAGTAC CTTACAATCC ACAAAGCCAA

GGAGTAGTGG AAGCAATGAA TCACCACCTA AAAAATCAGA TAAGCAGAAT

TAGAGAGCAG GCAAACACAG TAGAAACAAT AGTACTAATG GCAGTTCATT

GCATGAATTT TAAAAGGAGG GGAGGAATAG GGGATATGAC CCCAGCAGAA

AGACTAATCA ATATGGTCAC TGCAGAACAG GAAATACAAT TCCTCCAAGC

AAAAAATTCA AAATTACAAA ATTTTCGGGT CTATTTCAGA GAAGGCAGAG

ATCAGCTGTG GAAAGGACCT GGGGAACTAC TGTGGAAGGG GGACGGAGCA

GTCATAGTCA AGGTAGGGGC TGACATAAAA ATAATACCAA GAAGGAAAGC

TAAGATCATC AAAGACTATG GAGGAAGGCA AGAGATGGAT AGCGGTTCCA

ACTTGGAGGG TGCCAGGGAG GATGGAGAGG TGGCATAGCC TTATCAAGTA

TCTAAAATAC AGAACAGGAG ATCTAGAGAA GGTGTGCTAT GTTCCCCACC

ATAAGGTGGG ATGGGCGTGG TGGACTTGCA GCAGGGTAAT ATTCCCATTA

AAAGGAGAAA GTCATCTGGA GATACAGGCA TACTGGAACC TAACACCAGA

AAAAGGATGG CTCTCCTCCT ATTCAGTAAG ACTAACTTGG TATACAGAAA

AATTCTGGAC AGATGTTACC CCAGACTGTG CGGACTCCCT AATACATAGC

ACTTATTTCT CTTGCTTTAC GGCAGGCGAA GTAAGAAGAG CCATCAGAGG

GGAAAAGCTA TTATCCTGCT GCAACTACCC CCAAGCCCAT AAGTACCAGG

TACCGTCACT CCAGTTTCTG GCCTTAGTGG TAGTGCAACA AAATGGCAGG CCCCAGAGAG ACAATACCAC CAGGAAACAG TGGCGAAGAA ACTATCGGAG AGGCCTTCGA GTGGCTAGAC AGGACGGTAG AAGCCATAAA CAGAGAGGCA GTGAACCACC TGCCCCGAGA GCTTATTTTC CAGGTGTGGC AAAGGTCCTG GAGATACTGG CATGATGAAC AAGGAATGTC AATAAGTTAC ACAAAGTATA GATATTTGTG CCTAATGCAG AAAGCTATGT TCATACATTC TAAGAGAGGG

TGCACTTGCC TGGGGGGAGG ACATGGGCCG GGAGGATGGA GATCAGGACC TCCCCCTCCT CCCCCTCCAG GTCTAGTCTA ATGACTGAAG CACCAACAGA GTCTCCCCCG GAGGATAGGA CCCCACCGAG GGAGCCAGGG GATGAGTGGG TAATAGAAAC CCTGAGAGAG ATAAAATAAG AAGCTTTAAA GCACTTTGAC CCTCGCTTGC TAATTACTCT TGGCAACTAT ATCTATGCTA GACATGGAGA CACCCTTGAA GGCGCCAGAG GGCTCATTAG GATCCTACAA CGAGCCCTCC TCTTGCACTT CAGAGCAGGA TGCGGCCGCT CAAGGATTGG TCAGCCCAGG GGACGAAATC CTTTATCAGC TATACCAACC CCTAGAGGCA TGCGATAACA AATGTTACTG TAAAAAGTGC TGCTACCATT GCCAGATGTG TTTTTTAAAC AAGGGGCTCG GGATATGGTA TGAACGAAAG GGCAGAAGAA GAAGAACTCC GAAGAAAACT AAGGCTCATT CGTCTTCTGC ATCAGACAAG TGAGTAAGAT GTGTGGTAGG AATCAACTAT TTGTTGCCAG CTTGCTAGCT AGTGCTTGCT TAATATATTG CGTCCAATAT GTGACTGTTT TCTATGGCGT GCCCGTGTGG AGAAATGCAT CCATTCCCCT CTTTTGTGCA ACTAAAAATA GAGATACTTG GGGAACCATA CAGTGCTTGC CAGACAATGA TGACTATCAG GAAATAGCTT TAAATGTGAC AGAGGCCTTC GACGCATGGA ATAATACAGT AACAGAACAA GCAGTAGAAG ATGTCTGGAG TCTATTTGAG ACATCAATAA AACCATGCGT CAAACTAACA CCCTTATGTG TAGCAATGCG TTGTAACAGC ACAACTGCAA AAAACACAAC CTCCACACCA ACAACCACCA CAACAGCAAA CACAACAATA GGAGAGAATT CTTCATGCAT ACGCACAGAC AACTGCACAG GGTTGGGAGA AGAAGAGATG GTCGACTGTC AGTTCAATAT GACAGGATTA GAGAGGGATA AGAAAAAACT ATATAATGAA ACATGGTACT CAAAAGATGT AGTCTGTGAA TCAAATGACA CCAAGAAAGA GAAAACATGT TACATGAACC ACTGCAACAC ATCAGTCATC ACAGAGTCAT GTGACAAGCA CTATTGGGAT ACTATGAGGT TTAGATATTG TGCACCACCG GGTTTTGCCC TGCTAAGATG CAATGATACC AATTATTCAG GCTTTGAGCC CAATTGTTCT AAGGTAGTAG CTGCTACATG TACAAGGATG ATGGAAACGC AAACCTCCAC TTGGTTTGGC TTTAATGGCA CCAGGGCAGA AAATAGAACA TATATCTATT GGCATGGTAG GGATAATAGA

ACCATCATTA GCTTAAACAA GTTTTATAAT CTCACCGTAC ATTGTAAGAG

GCCAGGAAAC AAGACAGTTG TACCAATAAC ACTCATGTCA GGGTTAGTGT

TTCACTCCCA GCCAATCAAT AGAAGACCCA GGCAAGCATG GTGCTGGTTC

AAAGGCGAGT GGAAGGAAGC CATGAAGGAG GTGAAGCTAA CCCTTGCAAA

ACATCCCAGG TATAAAGGAA CCAACGACAC AGAAAAAATT CGTTTTATAG

CGCTAGGAGA ACGCTCAGAC CCAGAAGTGG CATACATGTG GACTAACTGC

AGAGGAGAAT TTCTCTACTG CAATATGACT TGGTTCCTCA ATTGGGTAGA

AAACAGAACG AATCAGACAC AGCACAATTA TGTGCCATGC CATATAAAGC

AAATAATTAA TACCTGGCAC AAGGTAGGGA AAAATGTATA TTTGCCTCCT

AGGGAAGGAC AGTTAACCTG CAACTCTACA GTGACCAGCA TAATTGCTAA

CATTGACGGA GGAGAGAACC AGACAAATAT TACCTTTAGT GCAGAGGTGG

CAGAACTATA CCGATTAGAA TTGGGGGATT ATAAATTGAT AGAAGTAACA

CCAATTGGCT TTGCACCTAC ACCAGTAAAA AGATACTCCT CTGCTCCAGT

GAGGAATAAA AGAGGTGTAT TCGTGCTAGG GTTCTTAGGT TTTCTCACGA

CAGCAGGAGC TGCAATGGGC GCGGCGTCCT TGACGCTGTC GGCTCAGTCT

CGGACTTTAT TGGCCGGGAT AGTGCAGCAA CAGCAACAGC TGTTGGACGT

GGTCAAGAGA CAACAAGAAA TGTTGCGACT GACCGTCTGG GGAACAAAAA

ATCTCCAGGC AAGAGTCACT GCTATCGAGA AATACTTAAA GGACCAGGCG

CAACTAAATT CATGGGGATG TGCGTCTAGA CAAGTCTGCC ACACTACTGT

ACCATGGGTA AATGACACCT TAACGCCTGA TTGGAACAAC ATGACATGGC

AGGAATGGGA GCAACGAATC CGCAACCTAG AGGCAAATAT CAGTGAAAGT

TTAGAACAGG CACAAATCCA GCAAGAAAAG AACATGTATG AACTACAAAA

ATTAAATAGC TGGGATGTTT TTGGCAACTG GTTTGATTTA ACCTCCTGGA

TCAAATATAT TCAGTATGGA GTTTATATAG TAGTAGGAAT AATAGTTTTA

AGAATAGTAA TATATGTAGT ACAAATGTTA AGTAGACTTA GAAAGGGCTA

TAGGCCTGTT TTCTCTTCCC CCCCCGCTTA CTTCCAACAG ATCCATATCC

ACAAGGACCG GGAACAGCCA GCCAGAGAAG AAACAGAAGA AGACGTTGGA

AACAGCGTTG GAGACAATTG GTGGCCCTGG CCGATAAGAT ATATACATTT

CCTGATCCGC CAGCTGATTC GCCTCTTGAA CAGACTATAC AACATCTGCA

GGGACTTACT ATCCAGGAGC TTCCAGACCC TCCAACTAAT CTCCCAGAGT

CTTCGGAGAG CATTGACAGC AGTCAGAGAC TGGCTGAGAT TTAACACAGC CTACCTGCAA TATGGGGGCG AGTGGATCCA AGAAGCGTTC CGAGCCTTCG

CGAGGGCTAC GGGAGAGACT CTTACAAACG CCTGGAGAGG CTTCTGGGGC

ACACTGGGAC AAATTGGGAG GGGAATACTT GCAGTCCCAA GAAGGATCAG

GCAGGGGGCA GAAATCGCCC TCCTGTGAGG GACGGCGGTA TCAACAGGGA

GATTTTATGA ATACCCCATG GAGAGCCCCA GCAGAAGGGG AGAAAGGCTC

GTACAAGCAA CAAAATATGG ATGATGTAGA TTCAGATGAT GATGACCTAG

TAGGGGTCCC TGTCACACCA AGAGTACCAT TAAGAGAAAT GACATATAGG

TTGGCAAGAG ATATGTCACA TTTGATAAAA GAAAAGGGGG GACTGGAAGG

GCTGTATTAC AGTGATAGGA GACGTAGAGT CCTAGACATA TACTTAGAAA

AGGAAGAGGG AATAATTGGA GACTGGCAGA ACTATACTCA TGGACCAGGA

GTAAGGTATC CAAAGTTCTT TGGGTGGTTA TGGAAGCTAG TACCAGTAGA

TGTCCCACAA GAGGGAGATG ACAGTGAGAC TCACTGCTTA GTGCATCCAG

CACAAACAAG CAGGTTTGAT GACCCGCATG GAGAAACATT AGTTTGGAGG

TTTGACCCCA CGCTAGCTTT TAGCTACGAG GCCTTTATTC GATACCCAGA

GGAGTTTGGG TACAAGTCAG GCCTGCCAGA GGATGAATGG AAGGCAAGAC

TGAAAGCAAG AGGGATACCG TTTAGCTAAA AACAGGAACA GCTATACTTG

GTCAGGGCAG GAAGTAACTA ACAGAAAACA GCTGAGACTG CAGGGACTTT

CCAGAAGGGG CTGTTACCAG GGGAGGGACA TGGGAGGAGC CGGTGGGGAA

CGCCCTCATA CTTTCTGTAT AAATGTACCC GCTACTCGCA TTGTATTCAG

TCGCTCTGCG GAGAGGCTGG CAGATTGAGC CCTGGGAGGT TCTCTCCAGC

ACTAGCAGGT AGAGCCTGGG TGTTCCCTGC TAGACTCTCA CCAGTGCTTG

GCCGGCACTG GGCAGACGGC TCCACGCTTG CTTGCTTAAA AGACCTCTTA

ATAAAGCTGC CAGTTAGAAG CA