MOLECULAR CLONING AND CLONES OF HUMAN B LYMPHOTROPIC VIRUS (HBLV)

Abstract: A clone of Human B Lymphotropic Virus (HBLV), pZVH14 has been isolated (from the peripheral blood leukocytes of six individuals) and characterized. The clone is obtained from nucleic acids extracted from purified virus, and contains a 9.0 Kb HindIII fragment of HBLV. HBLV contains a double-stranded DNA genome and is morphologically similar to some members of the Herpes family. The clone is lymphotropic and selectively infects freshly isolated human B cells, where it induces the appearance of characteristic large refractile mononucleated or binucleated host cells.

Description: MOLECULAR CLONING AND CLONES OF HUMAN B LYMPHOTROPIC VIRUS (HBLV) A new DNA virus, designated Human B Lymphotropic Virus (HBLV), has been isolated from the blood leukocytes of patients with lymphoproliferative disorders. While the virus belongs morphologically to the Herpes family of viruses, HBLV, as shown below, this virus has not been previously characterized. HBLV is associated with some malignancies - ±n, " AIDS and non-AIDS patients, but is distinctly different than Human T-cell Lymphotropic Virus Type III (HTLV-III), the causative agent of AIDS. HBLV contains a large double stranded DNA genome, and selectively infects B cells; HTLV-III, on the other hand, contains a single stranded RNA genome, and selectively infects and is cytolytic for T cells. The nucleocapsid of the HBLV virus is of icosahedral symmetry with 162 capsomeres, and is enveloped in a lipid membrane. The outer surface of the viral envelope is covered with short spikes. The diameter of the enveloped virion is approximately 180 nm; the nucleocapsid is approximately 100 nm in diameter. The space between the capsid and the envelope, 35-40 nm, is filled with amorphous material. The nucleoprotein core or nucleoid is approximately 65 nm in diameter, and is occasionally rod-shaped or asymmetric. 1 Infection of primary cells or of cord blood cells 2 produces characteristic large cells 4-10 days after 3 infection. These cells are 2-4 times the diameter of 4 small leukocytes, and exhibit cy opathogenic and 5 ' cytolytic changes after about one week in culture. The 6 nuclei of these cells is often highly convoluted, 7 containing mainly euchromatic chromatin and nucleoli 8 without remarkable features. Large numbers of virions 9 are released by most infected cells. 10 GENERAL DESCRIPTION OF THE INVENTION II. The present invention is the production of a IX molecular clone of Human B Lymphotropic Virus (HBLV) and 13 the use of tha-fc clone •. in diagnostic and therapeutic 14 _procedures. 15 A new Human B Lymphotropic Virus (HBLV) has been 1.6 isolated from the peripheral blood leukocytes of six 17 individuals: three Human T-cell leukemia virus type III IS (HTLV-III) seropositive patients with Acquired Immune 19 Deficiency Syndrome (AIDS)-related lymphoma, two HTLV- 20. III seropositive patients with angio-immunoblastic 21 " . lymphadenopathy, and one patient with acute lymphoblastic 22 leukemia. All six isolates are closely related by 23 antigenic and molecular analysis, and sera from all six 24 virus-positive patients react immunologically with each 25 virus isolate. In contrast, only four sera from more 26 than 200 randomly selected healthy donors were 27 seropositive. HBLV contains a large double stranded DNA 28 genome and is morphologically similar to some members of 29 the Herpes virus group. HBLV selectively infects 30 freshly isolated human B cells, where it induces the 31 appearance of characteristic large, refractile 32 mononucleated or binucleated cells containing nuclear and 1 cytoplasmic inclusion bodies. However, HBLV is distin- 2 guishable from all the known human and sub-human primate 3 Herpesviruses by host range, biological effect on infec- 4 ted cells, and by lack of antigenic or genomic 5 relatedness. 6 In one of the preferred embodiments of the present 7 invention, molecular clone pZVH14 is used in the 8 detection of early virus infection of umbilical cord 9 blood lymphocytes and spleen cells by in situ 10 hybridization in culture. 11 It is believed that the present invention is 12 capable of introducing genes into target cells of the 13 virus as a recombinant DNA virus vector system. 14 The ultrastructural characteristics of the HBLV 15 virus, as well as its morphogenesis, place the virus in 16 the family of Herpesviruses -- with similarities to and 17 differences from "any known members of the family. Immune 18 electron microscopic studies show that patients from whom 19 the virus has been isolated make highly specific 20 antibodies to both the viral envelope and to internal 21 components of the virus. Immunological, molecular, 22 biological, and host range studies indicate that the HBLV 23 virus has not been previously described. 2:4- Cultures of mononuclear cells from infected blood 2.5 samples develop significant numbers of characteristic 26 large cells 4-10 days after culture with primary cells or 27 cord blood cells. Electron microscopy analysis shows 28 that HBLV virus particles are present in large cells but 29 absent in small lymphocytes. The infected cells are 2-4 0 times the diameter of small lymphocytes and do not show 1 any initial obvious σytopathic changes. After one week 2 in culture, however, cytopathic and cytolytic changes are 3 readily observable. Specifically, the nuclei of 4 infected cells are often highly convoluted; chromatin is 5 mainly euchromatiσ and contained nucleoli without remarkable features. The cytoplasm displayed fairly large Golgi apparatus, vesicles of different sizes, prominent arrays of rough endoplasmic reticulum, and abundant mitochrondria. The general appearance of these cells is that of highly polymorphic proliferating blasts of lymphoid origin. Specific immunolabeling of extracellular virus occurs at the ultrastructural level using pre-absorbed patient's serum and an antiserum against human gamma globulin raised in goats, and labeled either with ferritin or with peroxidase. Large numbers of virions are released by most infected cells, and appear in tight clusters at the surface of the cells. Virtually all of the virions are labeled at their periphery. In some instances, the label penetrates into the virion, indicating that the envelopes of some of the virions are not intact and that some of the patient's serum contains * antibodies to internal components of the virus as well as to the viral envelope. The HLBV virus of the present invention is propagated by infecting human blood cord cells with HBLV, as is described in more detail in the Specific Disclosure. STATEMENT OF DEPOSIT The subject matter of this invention, molecular clone pZVH14, has been deposited in the American Type Culture Collection in Rockville, Maryland, under ATCC No. 40247, and after allowance of this application, will be maintained for a term of thirty (30) years or five (5) years after the last request for such deposit or for the effective life of the patent, whichever is longest. The l; deposit will be replaced if the culture mutates or 2 becomes nonviable during the term of the deposit. DESCRIPTION OF THE FIGURE The Figure is the Southern Blot analysis of Human B Lymphotropic Virus genomic DNA. UTILITY STATEMENT " The molecular clone of the present invention is capable of in situ hybridization with host cells infected with HBLV. This clone, therefore, is useful as a 0 diagnostic probe for HBLV-infected cells, as well as for 1 the detection of viral antigens or antibodies in blood 2 samples (using an immunofluorescence assay or any other 3 assay for antigens or antibodies which uses viral nucleic 4 acids). Molecular clone pZVH14 is also used in the 5 detection of early virus infection of umbilical cord 6 blood lymphocytes and spleen cells by in situ 77 hybridization in culture. 8: ' The present invention is also capable of 9 " - introducing genes into target cells of the virus as a 0 " recombinant DNA virus vector system. 1 SPECIFIC DESCRIPTION OF THE INVENTION 2 In general, one ^method of cloning the Human B 3 Lymphotropic Virus (HBLV) genome involves isolating 4 unintegrated viral DNA after infection of primary cells 5 or cord blood cells with the HBLV virus. The 6 unintegrated viral DNA is then cloned in a lambda phage 7 library and screened with viral cDNA. 1 Infected primary cells and cultured peripheral 2 cord blood cells produce HBLV virus and serve as the 3 principal producer for immunological assays used to 4 detect virus specific antigens and antibodies in human 5 sera. Cultures of infected cells are grown and 6 harvested, followed by extraction of low molecular weight 7 DNA from newly infected cells. This produces 8 unintegrated viral DNA. A cDNA library is formed uing 9 HBLV cDNA. This cDNA is then used as a probe for ICC assaying unintegrated viral DNA. Unintegrated linear IX DNA (provirus DNA) is then obtained, containing the HBLV 12 genome of the present invention. This DNA is then 13 digested in a suitable plasmid to form clone pZVH14. 14 Two elements of the above process are well known 15 recombinant DNA procedures: the DNA library and the 16 cDNA probe. The library " is formed by taking the total 17 DNA from the infected primary or peripheral, blood cells, IS cutting the DNA into fragments with a suitable 19 restriction enzyme, hybridizing the fragments to a 20 radiolabeled cDNA probe, joining the fragments to plasmid 21 vectors, and then introducing the recombinant DNA into a 22 suitable host. 23 The cDNA probe is an HBLV cDNA probe made from 24. double-banded HBLV mRNA. A short oligo-dT chain is 25 hybridized to the poly-A tail of the mRNA strand. The 26 oligo-dT segment serves as a primer for the action of 27 reverse transcriptase, which uses the mRNA as a template 2.8 for the synthesis of a complementary DNA strand. The 29 resuling cDNA ends in a hairpin loop. Once the mRNA 30 strand is degraded by treatement with NaOH, the hairpin 31 loop becomes a primer for DNA polymerase I, which 32 completes the paired DNA strand. The loop is then 33 cleaved by SI nuclease to produce a double-stranded cDNA 34 molecule. Linkers are then added to the double-stranded cDNA by using DNA ligase. The linkers are cut open with a restriction enzyme and the cDNA is inserted into a suitable plasmid cleaved with the same enzyme; the result is a cDNA-σontaining recombinant plasmid. As shown in Example 5, the molecular clone pZVH14 of the HBLV genome is useful as a template for radiolabeled RNA using T7 RNA polymerase, S-labeled dGTP, and unlabeled ribotriphosphates. In the preferred embodiment of the present invention, supernatant 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 Beck an SW41 rotor at 4°C. The pellets are suspended up in TNE buffer (10 mM, Tris-Cl, pH 9, 100 mM NaCl:l mM EDTA), and extracted with PC19 (Phenol:Chlorofor :Iso- amyl alcohol; 50 mM Tris-Cl, pH 9:: 100:100:1:10 : : v:v:v:-v) • ' followed Chloroform:Isoamyl alcohol (24:1: :v:v). Enriched viral DNA is precipitated by adding 2 volumes of 95% ethanol. DNA is digested with Hindlll and cloned into the Bluescrib vector (commercially available from Vector Cloning Systems, CA). Several clones obtained after screening with labeled, enriched DNA were examined for specificity of hybridization to infected human umbilical cord blood cell DNA and by in situ hybridization to infected cells. Specific hybridization of HBLV clone ZVH14 to DNA from pelleted virus digested with Hindlll (Fig. 1, panel A) and EσoRI (Fig. 1, panel B). Extracellular virus is shown in lane 1, virus infected human umbilical cord blood cells in lane 2, and negative control DNA isolated from the skin of an AIDS patient in lane 3. Clone ZVH14 scored positive in these assays and did not hybridize to uninfected controls. The infected cell DNA shown in 1. 1'ane 2 is isolated after several rounds of cell free 2! virus transmission in human umbilical cord blood cells. 3 EXAMPLES 4 Example 1. Several DNA clones obtained from nucleic 5 acids extracted from purified virus obtained as described 6 above, were examined for specificity and for comparison 7. with other DNA viruses. One HBLV clone designated 83 pZVH14, which contained a 9.0 Kb Hindlll fragment, was 9 " used for these studies. Southern blot analysis showed 10 the presence of viral specific DNA in Hindlll and EcoRI 11 digests of DNA from both purified virus and HBLV-infected 12 ' human cord ' blood cells. In situ hybridization 13 " . experiments with the same probe also confirmed.that' these 14 sequences were confined to infected cells. 15 Example 2. Human B Lymphotropic Virus clone pZVH14 has 16 been restriction enzyme mapped, as follows: 17 ' Kb 1 .0 2.0 3. 0 4.0 5.0 6.0 7.0 8.0 9 .0 18 T3→. «_T7 21 6.3Kb 8 .2Kb 22 wherein B=BamHI; E=EcoRI; Xh=XhoI; H=HindIII; P=PstI, and 23 H ^ ^H____indicates viral fragments detected with 24 pZVH14 insert in infected cells and viral DNA 25 preparations using EcoRI. Example 3. Molecular probes specific for HSV-1, CMV, and EBV (Herpes Simplex Virus type 1, cytomegalovirus, and Epstein-Barr virus, respectively) were used for comparisons with HBLV. While each individual viral probe specifically hybridized to its homologous nucleic acids, HBLV was clearly distinct from these transforming human DNA viruses. Furthermore, the size of the HBLV genome was shown to contain a minimum complexity of llOkb-pair as determined by analysis of sucrose gradient purified viral DNA. This genome size, as well as other features, also distinguishes HBLV from DNA viruses of the adenovirus, polyomavirus, papovavirus, and papillomavirus groups. Despite morphological and other properties similar to some of the herpesviruses, HBLV appears to be a new human DNA virus. It_ is distinguishable from other viruses by biological properties and ' by a lack of immunological and genomic homology. HBLV is highly lytic in vitro, as are CMV, HSV, HVS, and HVA, but has a narrower host range than these viruses or EBV, being limited to a subset of B-cells. Example 4. Umbilical cord blood lymphocytes were co- cultured with AIDS patient's blood serum (cells). Characteristic large refractile cells appeared in the cord blood cultures. After six days in culture, the cells were pelleted and the supernatants were layered over 20% glycerol and spun in the centrifuge (Beckman SW41 or SW28) for 3 hours at 25,000-30,000 rpm. The pellets contained cell debris and virus particles. Phenol extraction of the pellet produced large amounts of viral nucleic acid. A double-stranded DNA clone, ZVH14, obtained from this nucleic acid preparation was used for - lO - ll specific hybridization detection of the virus in Southern 2. Blot experiments and in in situ hybridization 3 ~ experiments. 4 Example 5. In situ hybridization of HBLV-infected human 5; cord blood cells. Experiments were performed 6 utilizing: 35s-iabeled RNA probes as described in the 7 Specific. Disclosure. Clone pZVH14 of the HBLV genome 8 was used as a template for radiolabeled RNA using T7 RNA 9~ poly erase, 35s-iabeled dGTP, and unlabeled ICC ribotriphosphates. Less than one grain per cell was 11 observed in uninfected negative control cultures. Large 12 refractile cells characteristic of the infected cultures 13, were heavily labeled, indicating the expression of 14" abundant viral messages.

Claims: WHAT IS CLAIMED IS: 1. A Human B Lymphotropic Virus clone, pZVH14, corresponding to American Type Culture Collection Accession No. 40247. 2. In a method of cloning genomic sequences of a virus wherein viral DNA is cloned into a cloning vector, the improvement which comprises cloning viral DNA sequences from Human B Lymphotropic Virus. 3. The method of Claim 2, wherein said viral DNA is a 9.0Kb Hindlll DNA fragment. " . ' • * 4.. A method for testing DNA clones of Human B Lymphotropic Virus containing a 9.0 Kb Hindlll fragment which comprises examining by Southern Blot analysis. 5. A Human B Lymphotropic Virus clone with the following restriction enzyme map: Kb 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 T3-* 4-T7 Xh P Xh 6.3Kb 8.2Kb wherein B=BamHI; E=EcoRI; Xh=XhoI; H=HindIII; P=PstI, and W3KB tf Λ m WM indicates viral fragments detected with pZVH14 insert in infected cells and viral DNA preparations using EcoRI.

Inventors: GALLO ROBERT C, JOSEPHS STEVEN FRANCIS, SALAHUDDIN SYED ZAKI, WONG-STAAL FLOSSIE YEECHING, GALLO, ROBERT, C., JOSEPHS, STEVEN, FRANCIS, SALAHUDDIN, SYED, ZAKI, WONG-STAAL, FLOSSIE, YEECHING
Applicants: US COMMERCE, THE UNITED STATES OF AMERICA, AS REPRESENTED BY TH
Application year: 1987
Main classification: C12Q1
IPC codes: A61K39, C07K14, C12N7, C12N15, C12Q1, C12R1