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with the clinical state of the infected individual. J Clin Invest 1991;87:1462-6. Zarling J. Moran P. Grosmaire L. McClure J. Shriver K. Ledbetter J. Lysis of cell infected with HIV-I by human lymphocytes targeted with monoclonal antibody heteroconjugates. J Immunol 1988; 140:2609-13. Lotscher E. Steimer K. Capon D. Baentiger J. Jack H. Wabl M. Bispecifie antibodies that mediate killing ofcells infected with human immunodeficiency virus of any strain. Proc Natl Acad Sci USA 1991;88:4723-7. Willey RL. Smith DH. Lasky LA. et al.ln vitro mutagenesis identifies a region within the envelope gene of the human immunodeficiency virus that is critical for infectivity. J Virol 1988;62: 139-47. Dournon E. Rozenbaum W. Michon C. et al. Effects ofzidovudine in

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365 consecutive patients with AIDS or AIDS-related complex. Lancet 1988;2: 1297-302. Kim Y. Fung M. Sun N. Sun C. Chang N. Chang T. Immunoconjugates that neutralize HIV virions kill T cells infected with diverse strains of HlV-1. J ImmunoI1990;144:1257-62. laRosa OJ. Davide JP. Weinhold K. et al. Conserved sequence and structural elements in the HIV-I principal neutralizing determinant. Science 1990;249:932-5. Zarling J. Moran P. Haffar O. et al. Inhibition of HIV replication by pokeweed antiviral protein targeted to CD4+ cells by monoclonal antibodies. Nature 1990;347:92-5. Pincus S. Wehrly K. Chesebro B. Treatment of HI V tissue culture infection with monoclonal antibody-ricin A chain conjugates. J Immunol 1989; 142:3070-5.

Anthony Simmons, Yvonne Demmrich, Antonietta La Vista, and Kym Smith

Divisions of Medical Virology and Tissue Pathology. Institute ofMedical and Veterinary Science. Adelaide. Australia

Mink lung epithelial (NBL-7) cells were shown to be permissive for human herpesvirus 6 (HHV-6) by four independent methods of analysis: detection of infectious virus, viral antigens, viral DNA sequences, and herpesvirus particles. Infection was serially passaged, with minimal cytopathology, for several months demonstrating for the first time that a cell of epithelial origin can support HHV-6 replication.

Human herpesvirus 6 (HHV-6) is a recently discovered agent [I] that displays tropism for human CD4+ T cells [2]. It is associated with a number of clinical entities including exanthem subitum [3] and lymphadenopathy [4], and asymptomatic oral shedding of HHV-6 is highly prevalent among healthy adults [5]. The virus has been successfully propagated in vitro in human adult and umbilical cord lymphocytes [6], human T cell lines [7], thymocytes [8], and more recently human fibroblasts [9]. Certain HHV -6 strains may also replicate in other cell types such as Epstein-Barr virustransformed B lymphocytes [10] and cell lines ofmegakaryocytic or glial origin [11], but there have been no reports of growth ofHHV-6 in epithelial cells. A continuing search for other cell-culture systems able to support replication of HHV-6 has merit because biologic studies have so far been complicated by the highly cell-associated nature of infectivity of most strains in vitro, and observations made in cultured cells may assist in understanding the pathogenesis of HHV-6 infection.

Received 12 November 1991; revised 18 February 1992. Dr. A. Simmons. Division of Medical Virology. Institute of Medical and Veterinary Science. Frome Rd .. Adelaide SA 5000. Australia.

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© 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6601-0035$01.00

DNA sequence analysis has shown unequivocally that HHV-6 is most closely related to human cytomegalovirus [12]. Few cell lines are permissive for cytomegalovirus, which is most commonly isolated and propagated in human diploid fibroblasts. The mink lung epithelial cell line, NBL7, is the only nonprimate cell type that readily supports cytomegalovirus replication [13]. These cells, which are derived from trypsinized lungs of Aleutian mink (Mustela vison) fetuses, are vigorous, contact-inhibited cells with an epithelioid appearance in culture. In this study, we used NBL-7 cells (ATCC CCL64) to maintain HHV-6, strain Z-29, in serially passaged cultures.

Materials and Methods Virus. Experiments were done with strain Z-29, one of the earliest isolates ofHHV-6 [6). Viral stocks were grown in phytohemagglutinin (PHA)-stimulated cord blood lymphocytes and cultured in RPMI 1640 (10 6 cells/rnl) containing 10% fetal calf serum and 10 units/ml human recombinant interleukin-2 (Boehringer Mannheim, Mannheim, Germany). Infected cells were transferred to fresh cultures (ratio of infected to uninfected cells, I: 10) at weekly intervals. Stocks were stored in liquid nitrogen as viable cryopreserved 7-day infected cells (10 7 cells in l-ml aliquots) in 10% dimethyl sulfoxide. Anticomplement immunofluorescence (Ae/F). Infection of cells was monitored by ACIF [6], rather than direct immunofluo-

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ded in epoxy resin. Before examination, sections were doublestained with lead citrate and uranyl acetate.

Results Before infection, NBL-7 cells were grown to confluence in 25-cm 2 tissue culture flasks using Dulbecco's MEM . Cultures were seeded with 0.5-mt samples (- 5 X 105 TClDso) of freshly thawed viral stock, maintained in medium containing I % fetal calf serum, and examined daily by phase-contrast microscopy for the presence of cytopathic changes. At weekly intervals, cells were harvested by exposure to 0.1 % trypsin/GS mM EDTA and passaged (I: 10) to fresh confluent NBL- 7 monolayers. At each passage, cell smears were examined by ACIF for viral antigens and un infected cultures were processed in parallel throughout. Although cytopathic effect was minimal, occasional clusters (typically I0/25-cm 2 flask) of enlarged, sometimes multinucleated cells were observed in cell monolayers exposed to virus as early as passage 2 (figure IA) . These foci, which were not seen in uninfected control cultures, did not progress to form plaques, but rather, swollen cells were shed into the culture medium where they tended to accumulate until the culture was passaged. ACIF results were inconclusive at this time. Specific cytoplasmic and nuclear fluorescence , indicative of viral antigen production , was unequivocal after blind passage 7, and at this stage infection was confirmed by two additional methods of analysis. First, intracellular herpesvirus-like particles were seen in whole cell preparations examined by electron microscope (figure I B); second, HHV-6 specific DNA sequences were detected in celllysates by slot blot analysis. The proportion of ACI~ cells increased rapidly, stabilizing at - 90% by passage I I (figure I C). Despite this high level of infection, maintaining selected cultures for 1421 days did not result in visible cytopathic effect. The amount of infectious virus produced by NBL-7 cultures,

B Figure 1. A, Phase contrast photomicrograph showing focus of ballooned cells in HHV-6-infected NBL-7 monolayer. Foci were scarce and did not progress to plaque formation. B, High-power electron micrograph showing herpesvirus particle in cytoplasm of mink lung epithelial cell 7 serial passages after infection. Bar = 100 nm. C, Anticomplement immunofluorescence staining of NBL-7 cells for viral antigens II serial passages after infection with HHV-6 strain Z-29. Speckled cytoplasmic or nuclear fluorescence was present in >90%of cells. Neither cytoplasmic nor nuclear fluorescence was seen in cells from uninfected cultures.

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rescence, to avoid potential background staining caused by the presence ofB lymphocytes in cord blood preparations. Acetonefixed cell smears on glass slides were reacted first with heat-inactivated human serum containing antibodies to HHV-6. Specific antibody binding was detected by fixation ofguinea pig complement (prepared in the laboratory), followed by application of fluorescein isothiocyanate-conjugated anti-guinea pig C 3 (Organon Teknika, West Chester, PA). All reactions were allowed to proceed for 30 min at 37°C in a humidified environment, and PBS (pH 7.4) was used to dilute reagents and wash slides between steps. Viral titration. Infectious virus was quantified using an adaption of a standard procedure for determination ofTCIDso. Tenfold dilutions of test material were incubated with PHAstimulated cord blood lymphocytes in 24-well plastic tissue culture dishes (ICN Flow, Sydney; 106 cells/well). At least four replicates were tested at each dilution . After 7 days of incubation, cell smears from each well were examined by ACiF for viral antigen, and the TCiD so value was calculated by the method of Reed and Muench [14J. The titer of freshly thawed viral stock was typically 104 TCIDso/ml. Detection of viral DNA sequences. Riboprobes were generated from plasmid pH6Z-1 03 (gift of P. Pellet, Centers for Disease Control, Atlanta), which consists ofa 3.9-kb BamHI fragment ofHHV-6, Z-29, cloned into Bluescript M I r (Stratagene Cloning Systems, La 101la, CA). Cells were lysed in 0.4 M NaOHjO .1% Triton X-I 00. Supernatant samples (100 ~I) were also treated with 0.4 M NaOHjO.1 % Triton X-I 00 before filtration. Samples were slot blotted onto Zetaprobe membrane (BioRad Laboratories. Sydney) and hybridized overnight at 55°C in a total volume of 10 ml containing 3.3 X 108 dpm/eg 32P_la_ beled riboprobe in the presence of0.8 MNa+ and 50%deionized formamide. Filters were washed in 2X SSC (I X SSC contains 0.15 M sodium chloride and 0.015 M sodium citrate) containing 0.1 %SDS for 10 min at room temperature and twice more for 15 min at 60°C. Finally, two stringent washes were done at 70°C in 0.1 X SSCjO.1%SDS. Filters were exposed to x-ray film (XAR; Kodak, Melbourne) for 16 h. Electron microscopy. Cells were fixed in 2.5% glutaraldehyde, postfixed in osmium tetroxide, dehydrated, and embed-

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measured at passages II (5 X 103 TCID 50/ml) and 17 (1.25 X 104 TCID 501ml), was similar to the yield obtained from PH A-stimulated cord blood cells. Infection was highly cellassociated, judged by slot blot analysis (figure 2) and absence of detectable infectious virus « 10 TCID 5o/ml) in culture supernatants at passages II and 17.

Discussion

A

B

C

Figure 2. Detection of ceIl-associated HHV-6-specific DNA sequences in infectedcultures by slot blot hybridization. Slot A: lysateof 5 X 106 uninfected NBL-7 cells. Slot B: supernatant (I 00 ~I) of NBL-7 culture, seven serial passagesafter infection. Slot C: lysate of 5 X 105 cells from NBL-7 culture, seven serial passages after infection.

agents, including reovirus type 3, vesicular stomatitis virus, and mammalian type C retroviruses from an unusually wide range of hosts [16]. Mink lung epithelial cells are, to our knowledge, the first nonprimate cells and the first line of epithelial origin shown to support replication of HHV-6 in vitro . However, it is interesting to note that HHV-6 antigen has been observed in tubular epithelial cells of rejected kidneys [17] . Viral yield from NBL-7 cultures was modest, and further studies with HHV-6 strains other than Z-29 are required to determine the usefulness of these cells for producing viral stocks. However, the lack of cytopathic effect in cultures maintained for up to 3 weeks is of potential importance because in situ hybridization and immunohistochemical studies suggest that noncytopathic infection of salivary gland tissue is the basis of persistent excretion of HHV-6 in saliva [18]. Consequently, infected NBL-7 cultures may provide a useful in vitro system for studying some of the pathogenetic mechanisms responsible for HHV-6 persistence.

Acknowledgements We thank Charli Bayley for typing the manuscript and Mark Fitz-Gerald for photographic work.

References I. Salahuddin SZ. Ablashi DV. Markham PO, et al. Isolation of a new virus. HBLV. in patients with Iymphoproliferative disorders. Science 1986;234:596-601. 2. Lusso P. Markham PD. Tschachler E. et al. In vitro cellular tropism of human B-Iymphotropic virus (human herpesvirus-e), J Exp Med 1988; 167:1659-70. 3. Yamanishi K. Okuno T. Shiraki K. et al. Identification of human herpesvirus-6 as a causal agent for exanthem subitum. Lancet 1988;1:1065-7. 4. Eizuru Y. Minematsu T. Mimanishima Y. Kikuchi M, Yamanishi K. Kurata T. Human herpesvirus 6 in lymph nodes. Lancet 1989; I:40. 5. Harnett G. FaIT T. Pietroboni G. Bucens M. Frequent shedding of human herpesvirus 6 in saliva. J Med Virol 1990;30 : 128-30. 6. Lopez C. Pellett P. Stewart J. et al. Characteristics of human herpesvirus-6. J Infect Dis 1988 ;157:1271-3. 7. Tedder RS. Briggs M. Cameron CH. Honess R. Robertson D. Whittle H. A novellymphotropic herpesvirus. Lancet 1987;2:390-2. 8. Roffman E. Frenkel N. Replication of human herpesvirus 6 in thymocytes activated by anti-CD3 antibody. J Infect Dis 1991;164:617-8. 9. Luka J. Okano M. Thiele G. Isolation of human herpesvirus 6 from clinical specimens using human fibroblast cultures . J Clin Lab Anal 1990;4:483-6. 10. Ablashi DV. Salahuddin SZ. Josephs SF. Imam F. Laso P. Gallo RC. HBLV (or HHV-6) in human cell lines [letter]. Nature 1987;329:207. II. Downing RG. Scwankambo N. Serwadda D. et al. Isolation of human Iymphotropic herpesviruses from Uganda . Lancet 1987 ;2:390. 12. Efstathiou S. Gompels UA. Craxton MA. Honess RW, Ward K. DNA homology between a novel human herpesvirus (HHV-6) and human cytomegalovirus. Lancet 1988 ; I:63- 4.

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Four independent methods of assessment (detection ofinfectious virus, viral antigens, HHV -6-specific DNA sequences, and herpesvirus-like particles) indicated successful propagation ofZ-29 in NBL-7 mink lung epithelial cells . The TCID50 assay showed that infection could be passed back to PHA-stimulated cord blood lymphocytes from epithelial cells, and the amount of infectious virus recovered from NBL-7 cultures was similar to that found in viral stocks. In lymphocytes, Z-29 infectivity is highly cell-associated [6] and this was also the case for virus grown in NBL-7 cells. Further investigations are required to determine the fate of NBL-7 cells productively infected with HHV-6. In cord blood lymphocyte cultures, the Z-29 growth cycle is -5 days, with nucleocapsids not being seen before day 3 [15]. Replication ofHHV-6 in lymphocytes is associated with ballooning and degeneration of cells, whereas minimal cytopathic effect was induced in NBL-7 cultures incubated for up to 21 days after infection, despite the presence of viral antigens in >90% of cells and production of infectious virus particles . The relationship between viral replication and the sparse foci of swollen cells observed from passage 2 onward is uncertain. The appearance ofcells within these foci resembled the cytopathology induced by HHV-6 in lymphocytes and the cytopathic effect associated with plaque-forming herpesviruses, such as cytomegalovirus, in cultured cells. However, ACIF results were equivocal in all cultures sampled before passage 7, and the frequency with which apparent cytopathology was detected remained low and constant (typically -10 clusters/Z'S-cm? monolayer), in stark contrast to the marked increase in ACIP cells over time . It is notable that NBL-7 cells are permissive for human cytomegalovirus, herpes simplex virus, and numerous other

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13. MacKenzie O. Mclaren LC. Increased sensitivity for rapid detection of cytomegalovirus by shell vial centrifugation assay using mink lung cell cultures. J Virol Methods 1989;26: 183-8. 14. Reed U. Muench H. A simple method for estimating 50 percent endpoints. Am J Hyg 1938;27:493-7. 15. Black JB. Sanderlin KC, Goldsmith CS. Gary HE. Lopez C. Pellett PE. Growth properties of human herpesvirus-6 strain Z29. J Virol Methods 1989;26: 133-44.

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16. Henderson CJ. Lieber MM. Todaro GJ. Mink cell line Mulha (CCL64). Focus formation and the generation of "non-producer" transformed cell lines with murine and feline sarcoma viruses. Virology 1974;60:282-7. 17. Okuno T. Higashi K. Shiraki K. et al. Human herpesvirus 6 infection in renal transplantation. Transplantation 1990;49:519-22. 18. Fox JO. Briggs M. Ward PA. Tedder RS. Human herpesvirus 6 in salivary glands. Lancet 1990;336:590-3.

Persistence of Measles Antibody after Revaccination Division of Immunization. Center for Prevention Services. Centers for Disease Control. Atlanta. Georgia; Division of Virology. Food and Drug Administration. Bethesda. Maryland; Division ofEpidemiology. Bureau ofCommunicable Disease Control. Massachusetts State Department ofPublic Health. Boston

To evaluate persistence of measles antibody after revaccination, antibody levels were measured 6 years after revaccination of 40 hemagglutination-inhibition (HAl) antibody-negative students who had participated in a serosurvey in Massachusetts. Twelve subjects who had been HAl antibody-positive and were not revaccinated were included as a comparison group. Before revaccination, 7 revaccinees had no detectable plaque reduction neutralization (PRN) antibody (group I) and 33 had low levels of PRN antibody (group 2). Three weeks after revaccination, all in group 1 and 30 (90%) of 33 in group 2 had developed a fourfold or greater rise in PRN antibody. Six years after revaccination, all subjects had PRN-detectable antibody. However, 12 in group 2 (36%) had antibody titers ~1:120 compared with none in group 1 (P < .01). Persons without PRN antibody will respond to revaccination and maintain protective antibody titers. In contrast, persons with low levels of PRN antibody may respond initially to revaccination, but their antibody titers may fall again to low levels.

In 1989, a two-dose measles vaccination schedule was recommended in the United States [I]. The major reason for the second dose is to reduce the number of persons susceptible to measles because of a lack of response to the first vaccine dose. An additional benefit of a second dose might be to boost antibody titers in persons who responded to the initial vaccination but whose titers later decreased to low or undetectable levels. There are few data on the efficacy of a second dose of measles vaccine in the United States administered for either of these reasons. Some information is available from a statewide cluster sample serosurvey done in Massachusettsstudents in grades 6,10, and 12 in 1982 [2]. In that study, 98% of the 1871 students had a history of measles vaccination; Received I November 1991; revised 18 February 1992. All participants gave informed consent. Reprints or correspondence: Dr. Lauri Markowitz. Information Services. Center for Prevention Services. COCo Atlanta. GA 30333. The Journal of Infectious Diseases 1992;166:205-8 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6601-0036$01.00

87% had detectable measles antibody by hemagglutination inhibition (HAl) (screening dilution, I:5). Students negative by HAl were retested by a more sensitive plaque reduction neutralization assay (PRN) [3]. Ofthe 247 HAl-negative students tested, 90% had detectable antibody by PRN. Students in grades 6 and 10 who were seronegative by HAl were contacted in 1983 to participate in a revaccination study. Of the 160 eligible students, 113 participated. Of these, 16 (14%) had no PRN-detectable antibody before revaccination and the primary vaccine was presumed to have failed; 97 had low levels of PRN antibody. Overall, III (98%) developed fourfold or greater rises in HAl antibody after revaccination and 106 (94%) developed fourfold or greater rises in PRN antibody [4]. IgM antibody was also tested; 14 (88%) of those without PRN antibody were positive for IgM after revaccination compared with only I of 68 having PRN antibody [4]. These data suggest that >94% of persons without detectable HAl antibody, including those in whom the primary vaccine failed and those who lost detectable HAl antibody after successful vaccination, will respond to revaccination.

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Lauri E. Markowitz, Paul Albrecht, Walter A. Orenstein, Susan M. Lett, Trish J. Pugliese, and Dorothy Farrell

Replication of human herpesvirus 6 in epithelial cells in vitro.

Mink lung epithelial (NBL-7) cells were shown to be permissive for human herpesvirus 6 (HHV-6) by four independent methods of analysis: detection of i...
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