Proc. Natd. Acad. Sci. USA Vol. 89, pp. 10552-10556, November 1992 Medical Sciences Human herpesvirus 7 is a T-lymphotropic virus and is related to, but significantly different from, human herpesvirus 6 and human cytomegalovirus (chronic fatigue syndrome/T lymphocytes) Zwi N. BERNEMAN*, DHARAM V. ABLASHIt, GE LI*, MAUREEN EGER-FLETCHER*, MARVIN S. REITZ, JR.*, CHIA-LING HUNGO§, IRENA BRUS¶, ANTHONY L. KOMAROFFII, AND ROBERT C. GALLO*,** *Laboratory of Tumor Cell Biology and tLaboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; TPharmacia, Columbia, MD 21045; NMount Sinai School of Medicine, New York, NY 10029; and IlDepartment of Internal Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115 Communicated by Maurice Hilleman, August 6, 1992 ABSTRACT An independent strain (JI) of human herpes- for 1-2 hr at 370C with virus-containing culture supernatant. virus 7 (HV-7) was isolated from a patient with chronic Afterward, the cell lines were cultured in RPMI 1640 medi- fatigue syndrome (CFS). No s cant association could be um/2-10%o fetal calf serum, whereas the CBMC were grown established by seroepidemiology between HHV-7 and CFS. in RPMI 1640 medium/5-20%o fetal calf serum with or with- HHV-7 is a T-lymphotropic virus, infecting CD4+ and CD8+ out 10% (vol/vol) interleukin 2 (Advanced Biotechnologies, primary lymphocytes. HHV-7 can also infect SUP-T1, an Columbia, MD). Infection was assessed by the appearance of immature T-cell line, with variable success. Southern blot large cells, immunofluorescence, and EM. analysis with DNA probes scanning 58.8% of the human Immunofluorescence. Indirect immunofluorescence with herpesvirus 6 (HHV-6) genome and hybridizing to all HHV-6 human polyclonal and murine monoclonal antisera was done strains tested so far revealed homology to HHV-7 with only and uninfected cells on 8- or 10-well slides 37.4% ofthe total probe length. HHV-7 contains the GGGTTA on infected control repetitive sequence, as do HHV-6 and Marek's disease chicken (1). The following HHV-6 monoclonal antibodies (mAbs) herpesvirus. DNA sequencing of a 186-base-pair fragment of were used: 9A5D12 (p41), 12B3G4 (p135), 6A5G3 (gpll6/ HHV-7(JI) revealed an identity with HHV-6 and human cyto- 64/54), 2D6 (gp82/105), 7A2 (gp102) (9) (from Bala Chan- megalovirus of 57.5% and 36%, respectively. Oligonudeotide dran, University of Kansas Medical Center, Kansas City); primers derived from this sequence (HV7/HV8, HV10/HV11) H-JG 16 (gplSO), H-AR 9 (gpl90), and H-AR 1-5 (gpll0/60) amplified HHV-7 DNA only and did not amplfy DNA from (from Janos Luka, University of Nebraska Medical Center, other human herpesviruses, incuding 12 different HHV-6 Omaha); C-5 (p38/41) (10) (from Gary Pearson, Georgetown strains. Southern blot analysis with the p43L3 probe containing University, Washington). The nature of infected cells was the 186-base-pair HHV-7 DNA fragment hybridized to HHV-7 determined by double immunofluorescence as described for DNA only. The molecular divergence between human cyto- Fig. 1. megalovirus, on the one hand, and HHV-6 and HHV-7, on the Southern Blot Hybridization. High-molecular-weight DNA other, is greater than between HHV-6 and HHV-7, which, in from cells infected with the following HHV-6 strains was turn, is greater than the difference between HHV-6 strains. examined: GS (1), CO1, C02, C03, C05 (11), U1102 (2), Z29 This study supports the classification ofHHV-7 as an additional (4), OK (12), KF, BA (13), SF (14), and DA (15). DNA from member of the human f3-herpesviruses. cells infected with the other human herpesviruses-herpes simplex virus type 1, Epstein-Barr virus, varicella-zoster An additional lymphotropic herpesvirus, human herpesvirus virus, and HCMV-was also analyzed (16). After digestion 6 (HHV-6), was reported a few years ago by several labora- with HindIII or BamHI, Southern blot analysis was done as tories (1-4). HHV-6 productively infects T lymphocytes in described for Fig. 3. The HHV-6 probes (16-21) used are vitro (5) and in vivo (6). Recently, another T-lymphotropic listed in Table 1. herpesvirus, human herpesvirus 7 (HHV-7), was described PCR Amplification, Cloning, and Sequencing of HHV-7 by Frenkel et al. (7). Independently, we have isolated HHV-7 DNA. HHV-7 sequence and clone were obtained as follows: (strain JI) from peripheral blood mononuclear cells (PBMC) Initial PCR amplification was in a 100-iul reaction, containing of a patient with chronic fatigue syndrome (CFS) (8). We 2 pug of HHV-7-infected SUP-T1 cell DNA; 10 mM Tris HCI describe here the serological, immunological, and molecular (pH 8.3); 50 mM KCl; 1.5, 2.0, or 2.5 mM MgCl2; 0.01% characteristics of HHV-7(JI). We also show that HHV-7 has gelatin; 0.2 mM (each) dATP, dCTP, dGTP, and dTTP; 4 ILM a tropism for primary human T lymphocytes and for the SUP-T1 T-cell line. A sequence was obtained of HHV-7 Abbreviations: CBMC, cord blood mononuclear cells; CFS, chronic DNA, which shows a homology with HHV-6 and with human fatigue syndrome; HCMV, human cytomegalovirus; HHV-6 and cytomegalovirus (HCMV).tt This HHV-7 sequence enabled HHV-7, human herpesvirus 6 and 7, respectively; PBMC, peripheral us to establish molecular reagents for identification of the blood mononuclear cells; mAb, monoclonal antibody. §Present address: Advanced BioScience Laboratories, Kensington, virus.# MD 20895. **To whom reprint requests should be addressed at: Laboratory of MATERIALS AND METHODS Tumor Cell Biology, National Cancer Institute, National Institutes of Health, Building 37, Room 6A09, Bethesda, MD 20892. In Vitro Infection. Cell lines or phytohemagglutinin A-stim- ttThe sequences reported in this paper have been deposited in the ulated cord blood mononuclear cells (CBMC) were incubated GenBank data base (accession no. L03525 for HHV-7 and L03526 for HHV-6). #lThis work was presented, in part, at the Annual Meetings of the The publication costs of this article were defrayed in part by page charge Laboratory ofTumorCell Biology, Aug. 17,1990, and Sept. 3, 1991, payment. This article must therefore be hereby marked "advertisement" National Cancer Institute, Bethesda, MD, and at the 15th Inter- in accordance with 18 U.S.C. §1734 solely to indicate this fact. national Herpesvirus Workshop, July 9, 1991, Pacific Grove, CA. 10552 Downloaded by guest on October 2, 2021 Medical Sciences: Bernernan et A Proc. Natl. Acad. Sci. USA 89 (1992) 10553 Table 1. Southern blot analysis of HHV-7(JI) DNA with Table 2. Sequence of DNA oligonucleotides used HHV-6 probes Name Sequence HHV-6 Insert Hybridization Primer for PCR probe size, kb to HHV-7(JI) Source (ref.) HV3.1 5'-GCNCCNTAYGAYATYCAYTTC-3' pZVH14 8.7 - (16,17) HV3.2 5'-GCNCCNTAYGAYATYCAYTTT-3' pZVB70 22.3 + (0.1x SSC) (18,19) HV3.3 5'-GCNCCNTAYGAYATACAYTTC-3' pZVB9 11.0 - (18) HV3.4 5'-GCNCCNTAYGAYATACAYTTT-3' pZVB43 8.0 - (17,18) HV4.1 5'-RAANARNGTNGCRATNGGNGT-3' pZVB10 6.2 + (0.1x SSC) (18) HV4.2 5'-RTANARNGTNGCRATNGGNGT-3' pZVB12 6.0 - (18) HV4.3 5'-RAANARNGTNGCTATNGGNGT-3' pZVB54 5.5 - (18) HV4.4 5'-RTANARNGTNGCTATNGGNGT-3' pZVB15 3.4 - (18) HV7 5'-TATCCCAGCTGTTTTCATATAGTAAC-3' pZVB16 2.5 - (18) HV8 5'-GCCTTGCGGTAGCACTAGATTTTTTG-3' pZVB2 2.0 - (18) HV10 5'-CAGAAATGATAGACAGATGTTGG-3' pZVB85 1.9 (18) HV11 5'-TAGATTTTTTGAAAAAGATTTAATAAC-3' pHD5 5.5 - (20, 21) Probe for pSMD6a 5.6 - (21) hybridization pH6Z-101 6.9 + (0.5x SSC) P. Pellett HV1 5'-(GGGTTA)6-3' The pHD5 and pSMD6a probes were from R. Honess (National HV9 5'-CCTAATGAAGGCTACTTTGAAGTACAAATG-3' Institute of Medical Research, London), and the pH6Z-101 probe HV12 5'-AGAATTCTGTACCCATGGGCACATTTGTAC-3' was from P. Pellett (Centers for Disease Control, Atlanta). SSC N, any nucleotide (A, C, G, T); Y, pyrimidine nucleotide (C, T); concentrations in parentheses indicate the most stringent wash after and R, purine nucleotide (A, G). Based on regions of amino acid hybridization at which signal could still be detected. identity (see Fig. 4), degenerate oligonucleotide primers were syn- thesized for PCR analysis: HV3.1, HV3.2, HV3.3, and HV3.4 for the primers HV3.1, HV3.2, HV3.3, HV3.4, HV4.1, HV4.2, APYDIHF peptide motif in a sense orientation, and HV4.1, HV4.2, HV4.3, and HV4.4; and 5 units of Taq DNA polymerase. HV4.3, and HV4.4 for the TPIATLF/Y motif, in an antisense Forty cycles ofPCR amplification were done according to the direction. Localization ofthe oligonucleotides on the 43L3 sequence following program: 94°C for 1 min, 45°C for 2 min, 72°C for were as follows: HV7, 1-26 (sense); HV8, 186-161 (antisense); HV9, 2 min with an increase of 2 sec an 82-111 (sense); HV10, 48-70 (sense); HV11, 171-145 (antisense); per cycle, and additional and HV12, 132-103 (antisense). 7 min at the end at 72°C. The PCR products were analyzed as described elsewhere (22). The polyacrylamide gel piece con- RESULTS taining the 228-base-pair (bp) PCR product was cut out, and the amplified DNA was eluted out of the gel piece in 0.5 M Cell Tropism of HIHV-7(JI). Double immunofluorescence ammonium acetate/i mM EDTA at 37°C overnight. After showed that infected CBMC are predominantly T lympho- phenol/chloroform extraction and ethanol precipitation, the cytes (Fig. 1). Infected cells, as determined by immunoflu- amplified fragment was redissolved in distilled water, treated orescence with patient's serum, showed the following char- with T4 polynucleotide kinase, extracted with phenol/ acteristics: 97% CD2+, 93% CD3+, 95% CD7+, 42% CD4+, chloroform, ethanol-precipitated, redissolved in distilled wa- 4% CD8+, 0%6 CD1+, 0%o CD19+, 0%6 membrane and cyto- ter, and blunt-end-ligated overnight at room temperature into plasmic-immunoglobulin+, 0o CDllb+, and 0%o CD16+.