CA1340403C

CA1340403C - Human b lymphotropic virus (hblv) isolation and products

Info

Publication number: CA1340403C
Application number: CA000543652A
Authority: CA
Inventors: Syed Zaki Salahuddin; Robert C. Gallo
Original assignee: United States Department of Commerce
Current assignee: United States Department of Commerce
Priority date: 1986-08-04
Filing date: 1987-08-04
Publication date: 1999-02-23
Anticipated expiration: 2016-02-23
Also published as: EP0318498A1; DE3784513D1; WO1988000980A1; ATE86308T1; IL83421A0; JPH02500324A; IL83421A; EP0318498A4; JP2559783B2; EP0318498B1; AU612330B2; AU7853487A; DE3784513T2

Abstract

A new human B-lymphotropic virus (HBLV) has been isolated. DNA, molecular clones, antigenic viral proteins and antibodies having specificity to the new virus have been prepared. A method for obtaining HBLV particles has the steps of growing a co-culture of infected patient blood with uninfected suitable target cells, isolating infected cells from the culture cell cloning, multiplying the infected cells by serial transmission, isolating a virus from the infected cell culture, and concentrating the virus. Various utilities of the new virus and products derived therefrom are described. Methods for detecting the presence of HBLV in a patient suffering from acquired immune deficiency syndrome; and for detecting antibodies specific to HBLV membrane antigens in human serum suspected of being infected with HBLV are also described.

Description

1340qO3 1 HUMAN B LYMPHOTROPIC VIRUS (HBLV) 4 A new Human B Lymphotropic Virus (HBLV) was isolated from the peripheral blood leukocytes of six 6 individuals: one HTLV-III seropositive patient with 7 AIDS-related syndrome; one HTLV-III seropositive patient 8 with angio-immunoblasted lymphoadenopathy; one patient 9 with dermatopathic lymphoadenopathy; a patient with mycosis fungoides; a patient with immunoblastic lymphoma;
11 and one patient with acute lymphoblastid leukemia. All 12 six isolates are closely related by antigenic and 13 molecular analysis, and sera from all six virus positive 14 patients reacted immunologically with each virus isolate. See Table 1. In contrast, only four sera from 16 more than 200 randomly selected healthy donors were 17 seropositive. HBLV contains a large double-stranded DNA
18 genome, and is morphologically similar to some members of 19 the Herpesvirus group. A detailed morphological analysis of HBLV is given below.
21 It selectively infects freshly isolated human B-22 cells, where it induces the appearance of characteristic 23 large, refractile mononucleated or binucleated ce~ls 1 containing nuclear and cytoplasmic inclusion bodies.
2 However, HBLV is distinguishable from all the known human 3 and sub-human primate Herpesviruses by host range, 4 biological effect on infected cells, and by lack of antigenic or genomic relatedness.
6 Despite morphological similarities, the host range 7 of HBLV contrasted with that of all members of the human 8 Herpesvirus group. For example, attempts to transmit the 9 virus to a number of T and B lymphoblastoid cells lines, and to a variety of other cell types, were unsuccessful.
ll In contract, Epstein-Barr Virus (EBC) infects most B-12 cells and some epithelial cells. Furthermore, other 13 Herpesviruses [e.g., Cytomegalovirus (CMV), Herpes 14 Simplex I and II (HSV) and Variocella-zoster Virus (VZV)]
infect a variety of cell types, often inducing cytopathic 16 effects. Other biological comparisons with EBV further 17 emphasized these differences. For example, no EBV
18 nuclear antigens were detected in HBLV-infected cord 19 blood mononuclear cells. This agent has been tentatively named Human B Lymphotropic Virus (HBLV) because of its 21 apparent uniqueness, its isolation from human tissues, 22 and its preference for B-lymphocytes.

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- 3a -SUMMARY OF THE lNV~NLlON 13 4 0 4 0 3 In one aspect of the invention, Human B Lymphotropic Virus (HBLV) virus particles are obtained by growing a co-culture of infected patient blood with uninfected suitable target cells; isolating the infected cells from the culture cell cloning; multiplying infected cells by serial transmission;
isolating virus from infected cell culture; and concentrating the virus.
In another aspect of the invention, a method of detecting the presence of HBLV in a patient suffering from acquired human deficiency syndrome is provided. The method comprises of co-culturing sera obtained from an AIDS patient with human B cells; purifying the HBLV by sucrose gradient method; and e~ ining for the presence of HBLV by enzy~e linked immunosorbent assay (ELISA), Western Blot and DNA dot analysis.
A further aspect of the invention provides for a method of detecting antibodies specific to HBLV membrane antigens in human serum suspected of being infected with HBLV. The method comprises (1) providing HBLV infected cells that have been a) washed in serum-free medium; b) separated from serum-free medium; and c) resuspended in serum-free medium. (2) incubating the resuspended HBLV infected cells with aliquots of each of: a) unknown serum sample; b) known HBLV antibody-negative serum; and c) known HBLV
antibody-positive serum, in order to obtain a separate test, a separate negative control and a separate positive control first reaction mixtures;
(3) separating the cells from each of the first reaction mixtures from supernatant fluid and ret~ining the cells; (4) washing the cells retained from the first reaction mixtures in serum-free medium; (5) resuspending the cells obtained in step (4) in serum-free medium; (6) separating the resuspended cells obtained in step (5) from supernatant fluid and ret~ining - 3b - 1340403 the cells; (7) incubating the retained cells in step (6) in serum free medium with fluorescein isothiocyanate (FITC)-conjuated anti-human immunoglobulin antibody in order to obtain a separate test, a separate negative control and a separate positive control second reaction mixtures; (8) separating the cells from each of the second reaction mixtures from supernatant fluid and retaining the cells; (9) resuspending cell retained in step (8) in serum-free medium;
(10) examining cells from each second reaction mixture of step (9) for membrane fluorescence; (11) comparing the fluorescence of test cells to the fluorescence of negative control and positive control cells; and (12) determining the presence or absence of antibodies to HBLV membrane antigens by the presence of or absence of fluorescent staining in the cells of step (10), respectively.

In a further embodiment, the invention provides a composition comprising antigenic proteins of an isolated human B Iymphotropic virus (HBLV) in a ph~ ce~ lly acceptable carrier, wherein said isolated human B Iymphotropic virus comprises a virus with:
(a) the morphology of a human herpes virus; and (b) a double-stranded DNA genome of about 170 Kb; and wherein genomic DNA from said isolated human herpes virus hybridizes under stringent conditions with nucleic acid of molecular clone ZVH14 (ATCC Accession No. 40,247), DESCRIPTION OF THE FIGURES
Figure 1 is the peripheral blood leukocytes from a patient with AIDS-associated lymphoma (a) and HBLV-infected umbilical cord blood leukocytes in cell culture (b).

-3c-Figure 2 is an electron micrograph of a cultured cell producing HBLV: I is peripheral mononuclear blood cells, II is PHA-P stimulated umbilical cord blood mononuclear cells, A is HBLV infected acetone fixed cells, B is HBLV infected live cells expressing membrane fluorese using HBLV
antibody, and C is immunofluorescence of HBLV infected cells with serum lacking HBLV antibody.
Figure 3 is in situ hybridization of HBLV-infected human cord blood cells.
Figure 4 is an immunofluorescent analysis of HLBV-infected cells.
Figure 5 is a southern blot analysis of HBLV genomic DNA.
Figures 6-12 are gel electrophoresis patterns of proteins recognized by human sera against HBLV.

1 The present invention is the isolation of a new 2 virus, Human B Lymphotropic DNA Virus (HBLV), associated 3 with some malignancies in AIDS and non-AIDS patients. In 4 order to identify and isolate HBLV, fresh peripheral blood mononuclear cells from AIDS patients with 6 associated lymphoproliferative disorders were established 7 in cell culture as described in Figure 1. In the 8 cultures of eight patients, primary cell cultures 9 contained a small number of large, refractile mononucleated or binucleated cells which survive for 11 short periods of time. These cells frequently contained 12 intranuclear and/or intracytoplasmic incluster bodies.
13 Electron microscope examination (Fig. 2a) shows that 14 these cells were infected by an enveloped DNA virus, 200 nm in diameter. These large cells were also the only 16 ones in culture expressing viral antigens, as measured by 17 fixed cell indirected immunofluorescence assays (IFA).
18 All three virus-positive patients were homosexual males 19 (2 white and 1 black, between the ages of 35 and 44), who were seropositive for HTLV-III with AIDS-pneumoncytsis 21 pneumonia, with Kaposi's sarcoma, and with 22 undifferentiated B-cell lymphoma.
23 The presence of the unique large, refractile cells 24 suggested the need for further examination of patients demonstrating morphologically similar cells in fresh or 26 cultures tissues.
27 HBLV from all six patients could be transmitted to 28 freshly isolated human leukocytes from umbilical cord 29 blood, adult peripheral blood, bone marrow, and spleen (previously stimulated with PHA-A as described in the 31 Specific Disclosure). After ln vitro infection in the 32 large refractile cells, noted in primary cultures, 33 appeared within 2-4 days post infection (Fig. lb). This 34 cell eventually became the predominant cell in the 13gO403 1 culture surviving for an additional 8-12 days. During 2 this time other cells in culture rapidly died. As in 3 primary cell cultures, these large cells expressed viral 4 nucleic acids as shown by in situ hybridization (Fig. 3), and viral antigens as detected by IFA (Figs. 4a and b).
6 Virus production was confirmed by electron microscopy 7 (Fig. 2b). HBLV-infected cells were typed for surface 8 markers defined by specific monoclonal antibodies, and 9 were found to express antigens recognized by B-cell 10specific Leu-12, Leu-16, Bl, and B4 monoclonal 11 antibodies. These cells lacked T-cell markers as 12 measured by OKT-3, OKT-4, and OKT-8 monoclonal 13 antibodies.
14Molecular probes specific for HSV-1, CMV, and EBV
were used for comparisons with HBLV. While each 16 individual viral probe specifically hybridized to its 17 homologous nucleic acids, HBLV was clearly distinct from 18 these transforming human DNA viruses. Furthermore, the 19 size of the HBLV genome was shown to contain a minimum complexity of 110 Kb-pair as determined by analysis of 21 sucrose gradient purified viral DNA. This genome size, 22 as well as other features, also distinguishes HBLV from 23 DNA viruses of the adenovirus, polymavirus, papovavirus, 24 and papillomavirus groups.

-~ 1340403 2 The subject matter of this invention, a molecular 3 clone called pZVH14 has been deposited in the American 4 Type Culture Collection in Rockville, Maryland under ATCC
No. 40247, and at issuance of this application into a 6 patent, will be maintained for a term of thirty (30) 7 years from the date of deposit or five (5) years from the 8 date of the last request for such deposit or for the 9 effective life of this patent, whichever is longest. The deposit will be replaced if the culture mutates or 11 becomes nonviable during the term of the deposit.

13 Despite morphological and other properties similar 14 to some of the Herpesviruses, Human B Lymphotropic Virus (HBLV) appears to be a new human DNA virus. It is 16 distinguishable from other viruses by biological 17 properties and by lack of immunological and genomic 18 homology. HBLV is highly lytic in vitro, as are CMV, 19 HSV, HVS, and HVA, but has a narrower host range than these viruses or EBV, being limited to a subset of B-21 cells. It is possible that HBLV could indirectly cause 22 abnormalities in B-cells leading to malginancy in vivo.

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1 Even though in the preliminary studies HBLV was 2 associated in five (5) instances with HTLV-III/LAV sero-3 positive donors, other evidence indicates that it is not 4 exclusively an AIDS-associated agent. Not only did all HTLV-III seropositive patients have complicating lympho-6 proliferative disorders, but HBLV was also isolated from 7 a HTLV-III seronegative ALL patient. Furthermore, 8 preliminary seroepidemiological analysis has shown a 9 reactivity clearly dissociated from HTLV-III individuals.
Serological comparisons demonstrate the uniqueness 11 of HBLV. Immunofluorescence assay was developed 12 following techniques originally described for 13 Herpesviruses, and was used to analyse patients and 14 healthy control sera, and to monitor infected cells.
Sera from all six HBLV positive patients demonstrated an 16 IgG antibody titer to viral capsid antigens (>1:20). In 17 contrast, only 4 of the more than 200 sera from randomly 18 selected healthy donors were positive. The pattern of 19 immunofluorescent staining in fixed, infected cells varied from punctate nuclear staining to diffuse staining 21 of the entire cell (Fig. 4a). In live cells, the 22 staining was confined to the cell membrane either as a 23 partial ring or in a capped form (Fig. 4b). Uninfected 24 cord blood mononuclear cells were negative when tested with sera from the 6 HBLV positive patients. Sera from 26 these positive patients also contained antibody to EBV
27 and CMV. A careful comparison of the titers of antibody 28 to EBV, CMV, and HBLV yielded a distinct titer for HBLV
29 as compared to that for EBV and CMV. Furthermore, the reactivity to EBV was completely removed by adsorption 31 with disrupted, EBV-infected cells or with purified EBV, 32 without significantly affecting the antibody titer to 33 HBLV.

1340~0~

1Sucrose gradient purification of HBLV. Hepar-2 anized peripheral blood leukocytes or human umbilical 3 cord blood mononuclear cells are banded in Ficoll-Hypaque 4 and established in cell culture at 36~C following PHA-F
(5~ug/ml) stimulation for 48 hours. The cells are then 6grown in RPMI-1640 supplemented with 10~ fetal bovine 7 serum (heat inactivated, 56~C for 30 min.) and 5~s/ml 8 hydrocortisone. Frozen supernatants obtained from the 9 infected cells are thawed, collected in 250 ml tubes and 10spun at 3500 rpm in a Sorall GSA rotor at 5cC for 10 11 min. The clarified supernatants are transferred to SW28 12tubes and spun and pelleted at 17,000 rpm for 90 min. at 13 ~C. Pellets obtained are resuspended in 10 mM Tris-Cl 14pH 7.4, 10 mM NaCl, 1 mM EDTA (TNE) to a volume of 300 microliters and layered onto a 15-60% sucrose gradient 162nd spun in an SW41 rotor (Beckman) at 20,000 rpm for 30 17 min at 5~C. Fractions of 1 ml are collected from the top 18 of the gradient. Each fraction is diluted to 10 ml and 19spun and pelleted in an SW41 rotor at 17,000 rpm for 90 min. Pellets are resuspended in 300 microliters of TNE
21 and aliquots assayed (by ELISA and Western 31Ot) for the 22 presence of virus and for virus infectivity. Human B
23 Lymphotropic Virus is easily detected in ractions 4-9 24 with a peak in fractions 5-7 by both assays. Extraction of nucleic acids from each fraction shows the presence of 26 double stranded DNA in fractions 5-9 with a peak in 27 fraction 7. Virus is also detected by electron 28 microscopy in the SW41 gradient pellet as well.
29Virus purified from fresh unfrozen supernatants according to the above procedure is used for detailed 31 electron microscopy.
32Ali~uots of the sucrose gradient fractions can be 33 assayed for the presence of HBLV by DNA dot blot analysis 34 using the pZVH14 9 Kb insert as a probe. The pZVHl4 * trade-mark 1 molecular clone may be obtained from the American Type 2 Culture Collection under Accession No. 40247.
3 The immunofluorescence assay and Western Blot 4 assay are the preferred assays for detecting HBLV
infection and HBLV antibodies in a variety of hemato-6 poietic malignancies, including B-cell lymphomas of both 7 AIDS and non-AIDS origin. The presence of HBLV
8 antibodies is elevated in the following disease groups, 9 but the invention is not intended to be limited to these specific diseases:
11 Burkitts lymphomas;
12 Hodgkin's disease;
13 A newly described infectious disease 14 syndrome similar to that seen in Lake Tahoe characterized as an "acute mononucleosis-like syndrome" in adults; and 16 ALL as diagnosed in children of Caribbean 17 and African origin.

18 HBLV Virus Propagation. Infection of human 19 umbilical cord blood or peripheral blood mononuclear cells is conducted by cell-free transmission as follows:

21 (1) Fresh blood samples are diluted 1:1 22 with RPMI-1640 and spun (and banded) on a Ficoll 23 gradient.

24 (2) The banded mononuclear cells are washed and put into culture in the presence of PHA-P (5~g/ml) 26 and HC (5~g/ml) in 20% FCS and RPMI-1640.

27 (3) After 24 hours, polybrene (2jug/ml) is 28 added to the culture and after 48 hours, the cells are 29 pelleted.

(4) A 1 ml aliquot of freshly harvested or frozen infected culture supernatant is added to the pellet and incubated at 37~C for 1 hour, with frequent agitation.

(5) Fresh medium [10% FCS and HC (5~g/ml) in RPMI-1640] is then added to the suspension, cultured, and incubated at 36~C.

(6) Within 2-10 days post infection, the characteristic enlarged refractile cells become visible. Supernatant is harvested at the peak of infection as measured by immunofluorescence and by visual observation of the culture for further transmission.
Cells infected by HBLV were also used to directly compare immunological cross-reactivities with other human and nonhuman primate Herpesviruses using specific monoclonal antibodies, hyperimmune sera, or sera from antibody positive control donors. As summarized in Table 1, all monoclonal antibodies to EBV, CMV, HSV, and hyperimmune sera to Rhesus CMV and African Green CMV, did not react with HBLV-infected cells. Furthermore, sera from several old world and new world primates many of which had antibodies to nonhuman primate Herpesviruses (including EBV-like viruses and CMV) did not show any cross-reactivity with HBLV-infected cells (Table 2).
Immunofluorescent Analysis of HBLV-Infected Cells. A modification of the indirect immunofluorescence assay developed by Henle, et al., Bacteriolo~Y, 91: 1248 (1966), was used for the detection of antibody to HBLV capsid atigens. For this assay, HBLV-infected or uninfected human cord blood mononuclear cells were washed 3 times for 10 minutes with PBS, resuspended _, . . .. . . .

- 11 - 13~0~03 1 in PBS, deposited on teflon coated slide, air dried, and 2 fixed in cold acetone for 10 minutes. Patient's sera 3 (heat inactivated at 56~C for 30 min. and clarified by 4 centrifugation) were added to the fixed cells, incubated in a humidity chamber at 37~C for 40 min , and washed with PBS, air dried, and stained with affinity purified 7 FITC conjugated anti-human IgG (H and L) for 40 min. The 8 cells were again washed as above, air dried, and mounted 9 with IF mounting solution. Larges cells with granular immunofluorescent staining, and cytoplasmic infected ll cells with HBLV as shown in Figure lb were assayed 5 days 12 post injection are shown in Figure 4. Small cells in the 13 background did not react with patient serum.
14 As is shown in Eig 4b, detection of viral membrane antigen HBLV infected as well as uninfected live 16 cells (non-fixed) were washed 3 times in serum-free medi-17 um, treated with patient's serum for 30 minutes at 4~C.
18 The cells were again washed, treated with affinity l9 purified FITC anti-human IgG for another 30 minutes, washed in medium again and examined for membrane 21 fluorescence. HBLV infected cells with surface patchy characteristics which were treated with patient serum fluorescence aftermembrane large cells - immunostaining is not shown.

Southern Blot Analysis of HBLV Genomic DNA.
Supernatants fluid from HBLV infected umbilical cord blood cells is layered onto 20% glycerol cushions and pelleted by centrifuging at 25,000 rpm for 3 hr. in a Beckman SW41 rotor at 4~C. The pellets are suspended up in TNE buffer (10 mM, Tris-Cl, ph 9, 100 mM NaCl:1 mM
EDTA), and extracted with PCl9 (Phenl:Choloroform:Isoamyl alcohol; 50 mM Tris-Cl, pH 9:: 100:100:1:10 :: v:v:v:v) followed Choloroform:isoamyl alcohol (24:1::v:v).
Enriched viral DNA is precipitated by adding 2 volumes of 1 95% ethanol. DNA is digested with HindIII and cloned 2 into the Bluescrib vector (commercially available from 3 Vector Cloning Systems, CA). Several clones were 4 obtained after screening with labeled, enriched, DNA were examined for specificity of hybridization to infected 6 human umbilical cord blood cell DNA and by in situ 7 hybridization to infected cells. Specific hybridization 8 of H3LV clone ZVH14 to DNA from pelleted virus digested 9 with HindIII (Fig.5 , panel a) and EcoR1 (Fig. 5 , panel b). Extracellular virus is shown in lane 1, virus 11 infected human umbilical cord blood cells in lane 2, and 12 negative control DNA isolated from the skin of an AIDS
13 patient in lane 3. Clone ZVH14 scored positive in these 14 assays and did not hybridize to uninfected controls. The infected cell DNA shown in lane 2 is isolated after 16 several rounds of cell free virus transmission in human 17 umbilical cord blood cells.

19 Example 1. Fresh tissue sections from 3 patients were found to contain a low number of HBLV-infecteed cells.
21 One patient, a 40 year old Hispanic with a history of IV
22 drug use, was seropositive for both HTLV-I and HTLV-III, 23 and was diagnosed with AIDS-pneumoncystic pneumonia with 24 associated dermatophathic lymphadenopathy. Another was a 61 year old white male who received multiple blood 26 transfusions in conjunction with open heart surgery 4 27 years prior to death. This patient was seropositive for 28 HTLV-III and was diagnosed with immunoblastic 29 lymphadenopathy with some skin involvement. A third patient was a 16 year old black male diagnosed with acute 31 lymphocytic leukemia of the T-cell type. Unlike the 32 others, this patient was seronegative for HTLV-III.
* trade-mark -- 13 - 13~ 0 4 03 Primary peripheral blood mononuclear cell cultures from these patients also contained a small number of the unique cells which, upon close e~. inAtion were also found to be infected by HBLV.

Example 2. A direct comparison of molecularly cloned sequences of the HBLV
genome [Salahuddin, et al., "Isolation of a New Human B Lymphotropic Virus (HBLV) from Patients with AIDS-Associated and Other Malignancies", Science, 234:596-601 (1986)] with the genomes of other Herpesviruses was also conducted. Several DNA clones obtained from nucleic acids extracted from purified virus were examined for specificity and for comparison with other DNA
viruses. One HBLV clone, designated pZVH14, which contained a 9.0 Kb HindIII
fragment, was used in these studies. Southern Blot analysis (Fig. 5) showed the presence of viral specific DNA in HindIII and EcoRI digests of DNA from both purified virus and HBLV-infected human cord blood cells. In situ hybridization experiments with the same probe also confirmed that these sequences were confined to the infected cells (Fig. 3).

Example 3. Monoclonal antibodies and hyperimmune sera prepared against human and simian Herpesviruses were tested for reactivity with HBLV infected cells by indirect immunofluorescence procedures as described.
Monoclonal antibodies to EBV and HCMB were used at 1:40 dilution;
HSV-l and II, VZV and HVS at a 1:10 dilution normal ascites fluid was used at 1:5 and 1:10 dilutions. Hyperimmune sera to African green and Rhesus monkey CMV were heat inactivated (50~C 30 min.) and clarified at 10,000 rpm and were used at 1:10 dilutions. In additon to the sera shown,human sera contAining antibodies to EBV, CMV, HSV-I and II, and VZV

- - 1340~03 1 also did not react with HLV infected cells. African 2 green monkey and Rhesus sera containing antibody to CMV
3 were also negative when tested with HBLV. Monoclonal 4 antibodies to EBV, and HCMV, and ascites fluid from normal mouse were gifts from Dr. Gary Pearson, School of 6 Medicine, Georgetown University, Washington, D.C. Mono-7 clonal antibodies to VZV and HVS were obtained from Dr.
8 Nancy Chang, Baylor College of Medicine, Houston, Texas, 9 and Dr. John Dahlberg, NCI, Bethesda, Maryland. HSV-I
and II monoclonal antibodies were purchased from Dupoint, 11 Boston, MA. Hyperimmune serum to purified African green 12 and Rhesus CMV were previously prepared in rabbits by Dr.
13 Ablashi.
14 Abbreviations used: HBLV, Human B Lymphotropic Virus; EBV, Epstein-Barr Virus; HCMV, Human Cytomegalo-16 virus; HSV, Herpes Simplex Virus; VZV, Varicella-Zoster 17 Virus; HVS, Herpes Virus Saimiri; VCA, viral capsid 18 antigen; EA, early antigen; MA, membrane antigen.
19 HBLV infected cord blood mononuclear cells were stained with an H3LV negative serum resulting in a 21 considerable number of large cells with no 22 immunofluorescence. The results are tabulated in Table 23 3.

24 Example 4. Serum from old world and new world primates were tested for antibody to HBLV by indirect immuno~luo-26 rescence as described.
27 Some sera from the old world primates were gifts 28 from Dr. P. Kanki, Harvard School of Public Health, 29 Boston, MA. All sera were heat inactivated at 56 C for 30 min., clarified by centrifugati.on before use. ~BLV-31 infected cord blood leu~ocytes, P3HR-l (an established 32 cell line expressing EBV-VCA), and Owl monkey kidney 33 cells infected by HSV-strain II were used for ~ .
13~0~03 1 comparisons. When infected cells showed cytopathic 2 effects, the cells were fixed in acetone and used for the 3 IFA.
4 Three owl monkeys and one cottontop marmoset were previously innoculated with HVS. Sera from these animals 6 possessed antibody to HVS late antigen which 7 cross-reacted with Herpesvirus ateles. The results are 8 tabulated in Table 2.

9 Example 5. In situ hybridization of HBLV-infected human cord blood cells. Experiments were performed 11 utilizing 35S-labeled RNA probes as described in the 12 Specific Disclosure. Clone pZVH14 of the HBLV genome was 13 used as template for radiolabeled RNA using T7 RNA
14 polymerase, 35S-labeled dGTP, and unlabeled ribotriphos-phates. Less than one grain per cell was observed in 16 uninfected negative control cultures. Large refractile 17 cells characteristic of the infected cultures were 18 heavily labeled, indicating the expression of abundant 19 viral messages.

Example 6. Two dimensional gel electrophoresis patterns 21 of proteins recognized by human sera against human B cell 22 lymphotropic virus (HBLV) are shown in Figures 6-12.
23 Human umbilical cord blood lymphocytes were infected with 24 HBLV and then labeled by incubation with 35S-methionine for periods of either 3 hours or 24 hours. H9 cells were 26 used as negative controls. The labeled cells were lysed 27 and the proteins immunoprecipitated according to 28 established procedures (Protein Data Bases, Inc., New 29 York). Spots seen on the gels of the lysates from infected cells but not seen on the control gels 31 represents candidate virus proteins arrayed in unique 32 virus specific patterns. These patterns serve as a '-' 13~040~

1 fingerprint which can specifically identify HBLV.
2 Included are Gels Nos. 16408 ( Figure 6), 16404 ( Figure 3 7), 16410 ( Figure 8), 16409 ( Figure 9), 16402 ( Figure 4 10), 16403 ( Figure 11), and 16405 ( Figure 12) .

C~oss-~eacLivity of Nonhumal1 ~ril1,ate Sera Virus Used to Infected Target Cells l,V l'~V ~ISV
~. Positive No. Positive No. Positive No. Tes~ed (VCA)/No. Tested No. Tested Serum Sources (Percent Posi1-ive) (Percent Positive) (Percent Positive) Old World Primates Chir panzee 0/5 (0) 5/5 (100%) 0/4 (0) Go~ 0/3 (~) 2/3 (66.6%) 0/3 (0) Orangutan 0/2 (0) l/2 (50%) 0/2 (0) Ba~oons 0/3 (()) 3/3 (100%) 0/3 (0) StumptaLL 0/2 (0) l/2 (50%) 0/2 (0) Rhesus 0/9 (0) 6/9 (66.6%) 0/7 (0) African Green 0/l0 (0) 6/l0 (60%) 0/l0 (0) New World Primates Squirrel Monkeys 0/l0 (0) 0/l0 (0%) 8/l0 (80) Owl Monkeys 0/6 (0) 0/6 (0~) 3/6 (50) Marmosets (common) 0/6 (0) 0/6 (0) 0/6 (0) Marmosets (~oLL~I~Lop) 0/3 (O) 0/3 (O) l/3 (33.3) C~
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Claims

1. A method of obtaining Human B Lymphotropic Virus (HBLV) virus particles by the following steps:
(a) growing a co-culture of infected patient blood with uninfected suitable target cells;
(b) isolating the infected cells from the co-culture;
(c) multiplying infected cells by serial transmission;
(d) isolating virus from infected cell culture; and (e) concentrating the virus.

2. A method of detecting the presence of human B lymphotropic virus (HBLV) in a patient suffering from acquired immune deficiency syndrome (AIDS) which comprises:
(a) co-culturing sera obtained from an AIDS patient with human B cells;
(b) obtaining specific fractions by sucrose gradient method;
(c) separating the fraction that will equilibrate at the point in the gradient that is equal to the density of HBLV; and (d) examining of said fractions for the presence of HBLV:
(i) by employing antibodies which recognize HBLV by enzyme linked immunoabsorbent assay (ELISA) and Western Blot; and (ii) employing pZVH14 by DNA dot analysis.

3. The method of claim 2, wherein examining for the presence of HBLV includes a Southern Blot analysis of the HBLV genomic DNA comprising:

(a) extracting DNA from the said cells;
(b) separating the resulting DNA fragments by gel electrophoresis;
(c) transferring separated DNA fragments to nitro cellulose;
(d) hybridizing the separated DNA fragment with probe ZVH14 (ATCC
Accession No. 40,247); and (e) subjecting said hybridized DNA fragments to autoradiography.

4. The method of claim 1, wherein the target cells are uninfected B lymphocytes from cord blood.

5. A method of producing HBLV virus proteins wherein in addition to the steps of claim 1, the virus proteins are isolated by gel electrophoresis from the virus-infected cell culture of step (d).

6. A first nucleic acid which hybridizes to a second nucleic acid, wherein said second nucleic acid comprises a nucleic acid from an isolated human B lymphotropic virus (HBLV) wherein said isolated human B lymphotropic virus comprises a virus with:
(a) the morphology of a human herpes virus; and (b) a double-stranded DNA genome of about 170 Kb; and wherein genomic DNA from said isolated human herpes virus hybridizes under stringent conditions with nucleic acid of molecular clone ZVH14 (ATCC Accession No. 40,247), wherein said first nucleic acid does not hybridize under stringent conditions with the nucleic acid of:

(i) Epstein-barr virus;
(ii) Human cytomegalovirus (CMV);
(iii) Herpes Simplex virus (HSV);
(iv) Varicella-Zoster virus (VZV); or (v) Herpes virus saimiri.

7. The nucleic acid of claim 6, wherein said first nucleic acid is molecular clone ZVH14 (ATCC Accession No. 40,247).

8. The first nucleic acid of claim 6, wherein said first nucleic acid is attached to a solid support.

9. The first nucleic acid of claim 6, wherein said first nucleic acid sequence is labelled.

10. A polypeptide encoded by said first nucleic acid of claim 6.

11. The polypeptide of claim 10, wherein said polypeptide is specifically bound by antibodies in human serum from a patient infected with an isolated human B lymphotropic virus (HBLV) wherein said isolated human B lymphotropic virus comprises a virus with:
(a) the morphology of a human herpes virus; and (b) a double-stranded DNA genome of about 170 Kb; and wherein genomic DNA from said isolated human herpes virus hybridizes under stringent conditions with nucleic acid of molecular clone ZVH14 (ATCC Accession No.
40,247).

12. A composition comprising antigenic proteins of an isolated human B
lymphotropic virus (HBLV) in a pharmaceutically acceptable carrier, wherein said isolated human B lymphotropic virus comprises a virus with:
(a) the morphology of a human herpes virus; and (b) a double-stranded DNA genome of about 170 Kb; and wherein genomic DNA from said isolated human herpes virus hybridizes under stringent conditions with nucleic acid of molecular clone ZVH14 (ATCC Accession No. 40,247).

13. A cell comprising said first nucleic acid of claim 6.

14. An antibody which binds specifically to an antigenic molecule from an isolated human B lymphotropic virus (HBLV) wherein said isolated human B lymphotropic virus comprises a virus with:
(a) the morphology of a human herpes virus; and (b) a double-stranded DNA genome of about 170 Kb; and wherein genomic DNA from said isolated human herpes hybridizes under stringent conditions with nucleic acid of molecular clone ZVH14 (ATCC
Accession No. 40,247).

15. A method of detecting HBLV in a biological sample comprising the steps of:
(a) contacting said biological sample with said antibody of claim 14 under hybridization conditions such that said antibody can specifically bind to human B lymphotropic virus antigenic molecules present in said biological sample whereby a complex is formed; and (b) detecting the presence or absence of said complex.

16. The method of claim 15, wherein said method comprises a Western Blot.

17. A method of detecting HBLV antigenic molecules in a biological sample comprising the steps of:
(a) contacting said biological sample with said first nucleic acid of claim 6 under stringent hybridization conditions to permit said first nucleic acid to hybridize to human B lymphotropic virus nucleic acids present in said biological sample whereby a complex is formed, and (b) detecting the presence or absence of said complex.

18. The method of claim 17, wherein said method is selected from the group consisting of a Southern Blot, a Northern Blot, and an in situ hybridization.