Replication of Type I Herpes Simplex Virus in Primary Cultures of Hairy Cell Leukemic Leukocytes Linda H. Pozner, Charles A. Daniels, MD, PhD, John A. Cooper, Harvey Jay Cohen, MD, Gerald L. Logue, MD, and Byron P. Croker, MD, PhD
The ability of leukemic leukocytes to support the replication of herpes simplex virus (HSV) was studied. Mononuclear leukocytes (MNL) from the peripheral blood of patients with a variety of lymphoid leukemias were isolated on Ficoll-Hypaque gradients and infected with HSV at a multiplicity of infection of 5 to 10. No virus growth was detected in cells from patients with chronic lymphocytic leukemia (9), acute lymphocytic leukemia (1), or lymphosarcoma cell leukemia (2). HSV replication did occur in hairy cell leukemic MNL from all of 4 patients studied. Maximal titers of 10' to 10'" PFU/ml occurred 1 to 7 days after incubation. By electron microscopy, herpesvirus particles were seen in the nuclei of these infected cells after 3 days of culture, but none was seen in the cells not exposed to virus. Fluorescent antibody mition confirmed the presence of HSV antigens in the nuclei of infected hairy cells. No difference in the adsorption or penetration of the virus was found with the various MNL studied. Productive infection of the cells thus appeared to depend on the ability of the leukocyte to support a later stage of infection, either uncoating or replication of the virus. (Am J Pathol 90:187-200, 1978)
A VARIE OF VIRUSES have been shown to be capable of replicating in human mononuclear leukocytes (MNL).1-7 The majority of these agents are able to productively infect freshly isolated MNL, and treatment of the cells with mitogen prior to infection markedly increases the amount of virus produced.3'6' Herpes simplex virus (HSV) is an exception, in that this agent will not productively infect freshly isolated MNL but will grow only in mitogen-stimulated leukocytes 1012 or continuous lymphoblastoid cell lines. lo Few investigators have studied the ability of leukemic leukocvtes to replicate virus. Gerber et al1 found that an avian myxovirus could grow in primary leukocyte cultures from patients with acute myelogenous leukemia, but little is known conceming the ability of human viruses to grow in freshly isolated leukemic lymphocytes. Since patients with leukemia have a propensity toward developing extensive and sometimes disseminated HSV infections,""'7 we reasoned that replication of this virus in From the Department of Pathology, Duke University Medical Center, and Department of Medicine, Division of Hematology, Veterans Administration Hospital, Durham, North Carolina. Supported in part by Grant CA 05634-16 from the National Cancer Institute and Grant DE 04609 from the National Institute of Dental Research, National Institutes of Health. Accepted for publication August 23, 1977. Presented in part at the Sixtv-first Annual Meeting of the Federation of American Societies for Experimental Biology, April 7, 1977, Chicago, Illinois. Address reprint requests to Dr. Charles A. Daniels, Department of Pathology, Duke Universitv 'Medical Center, Durham. NC 27i10.
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malignant leukocytes might play a role in the spread of infection. In.an effort to explore this hypothesis, we determined whether the lymphoid cells from the peripheral blood of patients with hairy cell (HCL), chronic lymphocytic (CLL), acute lymphocytic (ALL), and lymphosarcoma cell (LSCL) leukemia would support the replication of HSV. Materials and NWNMethodsPamb
Sixteen patients with malignant lymphoproliferative diseases and 4 normal subjects were studied, all of whom consented to have blood drawn for study. The normal subjects were classified as immune or nonimmune on the basis of the presence of anti-HSV antibodies in their heat-inactivated serum samples.u Nonimmune donors had no detectable neutralizing activity in their serum samples when they were tested at a 1:10 dilution, whereas immune subjects' serum samples had titers of greater than 1: 100. Nine patients, aged 56 to 74, had peripheral leukocyte abnormalities characteristic of CLL, including surface membrane immunoglobulin.ls. Four of these individuals were untreated; the remainder were receiving cytotoxic chemotherapy. Two patients with a history of nonHodgkin lymphoma had elevated peripheral blood leukocyte counts with a predominance of lymphoid cells characteristic of those seen in LSCL, including dense surface membrane immunoglobulin.2' Neither had received chemotherapy within 3 weeks of study. In addition, a 26-year-old patient was studied who had relapsed ALL with lymphoblasts comprising more than 95% of his peripheral blood leukocytes. Cells were also obtained from 4 patients with HCL, none of whom had received chemotherapy. Each patient initially presented with splenomegaly, anemia, thrombocvtopenia, and diffuse infiltration of the bone marrow by abnormal lymphoid cells. All 4 had peripheral blood mononuclear cells characteristic of HCL, including long filamentous projections by phase microscopy and the presence of intracellular tartrate-resistant acid phosphatase.22 Patient HCL-1, with a peripheral leukocyte count of 40,000 per cu mm and 99.5% HCL cells, had previously undergone splenectomy without demonstrable benefit. Patients HCL-2 and HCL-3 had been treated by splenectomy with partial resolution of anemia and thrombocvtopenia. Patient HCL-4 was untreated.
Vv1 md Antswwm HSV, strain CHR-HSV-3,23 was isolated from a patient with recurrent herpes labialis and identified as Type I HSV by virus neutralization tests using immune rabbit serum'4 and by plaque morphology on chick embryo fibroblasts.2 The virus, grown in human embryonic foreskin fibroblasts (Flow Laboratories, Bethesda, Md.), was assayed as plaqueforming units (PFU) on primary rabbit kidney (PRK) cells using an antibody overlay 0 and had a titer of 10"' to 10Z' PFU/ml. sH-labeled HSV was prepared by addition of 'Hthymidine (New England Nuclear, Boston, Mass.) to infected PRK cells according to the method of Notkins et al.'7 Human HSV immune serum samples were obtained from patients with recurrent herpetic infections. The serum samples, after heat inactivation at 56 C for 30 minutes, had neutralization titers of 1: 256 or greater as determined by the plaque reduction method."' Rabbit serum samples were obtained from adult New Zealand white rabbits either prior to immunization (normal rabbit serum samples) or 4 weeks after injection with PRK-grown HSV (immune serum samples)." HSV neutralization titers of normal rabbit serum samples were less than 1: 10, and those of immune serum samples were 1: 10,000.
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Isoation Iof Human Monnl Lesw cytgs
Ten to thirtv milliliters of venous blood was collected in heparinized (10 units/ml) svringes (Upjohn Co., Kalamazoo, Mich.) from each leukemic patient and each healthv donor. MNL were isolated from the whole blood samples by centrifugation on FicollHypaque gradients. The cells were washed twice in Eagle's minimal essential medium supplemented with 10% (v/v) fetal bovine serum, 50 jig/ml streptomvcin, and 100 units/ml penicillin G (EMBS),* and the concentration was adjusted to 2 X 10' cells/ml. The MNL from the leukemic patients were exposed to virus within 24 hours of isolation. When indicated in the text, normal MNL were either incubated with virus within 1 day of separation or exposed to phytohemagglutinin (PHA) and then infected with HSV. These normal cells were stimulated by incubating the MNL (2 X 10' cells/ml) with an equal volume of EMBS containing 2 pg/ml PHA (Wellcome Laboratories, Beckenham, England) for 3 days at 37 C and resuspended in fresh medium prior to infection. inftion of MNL
An equal volume of EMBS containing HSV (107- to 107 PFU/ml) was added to an MNL suspension (10"' cells/ml) (multiplicitv of infection of 5 to 10), and samples were incubated for 2 hours at 37 C. After washing twice, cells were treated for 60 minutes with human anti-HSV serum (1:4 dilution) to neutralize the unpenetrated virus and to synchronize the infection. MNL were washed again, dispensed into 1-mi portions containing 10' cells, and cultured at 37 C in a CO, incubator for intervals up to 7 days. At the times indicated, samples were removed and frozen at -70 C.
hb 4mSAssms MNL cultures were thawed and sonicated at 50 watts for 30 seconds with a Sonifier Cell Disrupter (Model W135, Heat Systems Ultrasonics, Inc., Plainview, NY). Cell debris was removed by centrifugation (300 X g for 10 minutes), and the supernatants wer-e assayed for infectivity. When virus replication in the leukocytes was demonstrated, i.e., an increase in virus titer of 0.3 log1, PFU/ml above the Day 0 value, the identity of the agent as Type I HSV was established by virus neutralization tests using immune rabbit serum 24 and by plaque morphology on chick embryo fibroblasts.2u In addition to the above, the number of infected cells was quantitated by an infectious center assay.5 Following treatment with antiviral serum and washing, the HSV-exposed MNL were adjusted to 10' cells/ml. One-tenth milliliter of serial 10-fold dilutions of each sample were plated in duplicate onto PRK monolayers. Each dish was overlaid with 4 ml of EMBS containing 2% (w/v) methvl cellulose (Methocel), and the plaques were counted after 3 days. po i Aup VAm 'H-labeled virus (200,000 cpm/ml) was incubated for 2 hours at 4 C with an equal volume of medium containing 2 X 10' MNL. Cells were washed three times, and the radioactivitv associated with the MNL was determined using a Packard Tricarb Scintillation Spectrophotometer (Packard Instruments, Chicago, Ill.). Adsorption studies were also performed by assessing the amount of virus depleted from the medium by the MNL.' HSV (2 X 10' PFU) was incubated with a suspension of 2 X 10' MNL or with medium alone (control). After 2 hours at 4 C, cells were centrifuged, and residual virus in the supematants was assaved for infectivity and compared with the control. * Unless otherwise stated, all washing procedures and dilutions of cells. virus. and antisenrms sere done in E%1BS.
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Floecn Antbody Stdies Infected and uninfected MNL were examined for HSV antigens by an indirect fluores-
cent antibody (FA) method. Rabbit serum samples. obtained as described above. were absorbed with uninfected human MNL and diluted 1: 10 in phosphate-buffered (pH 7.3) sodium chloride solution (PBS) containing 4%5 (w v) bovine serum albumin (BSA). Goat antirabbit IgG (heavy chain specific) conjugated with fluorescein isothiocyanate (FITC) (Cappel Laboratories, Downington, Pa.) was absorbed with human 5-globulin conjugated to Sepharose 4B ° and diluted 1 :2 in PBS-BSA. After 0. 1. 3. and 3 days of culture. cells were fixed to glass slides by spinning at 260 X g for 10 minutes on a cytocentrifuge (Shandon Southern Instruments, Sewickley. Pa.). The air-dried slides w-ere washed in PBS. fixed in acetone for 5 minutes at 25 C, and washed again in PBS. Each slide was then treated for 30 minutes with either normal or immune rabbit serum. Follow ing w-ashing in PBS. each slide was reacted with the FITC-conjugated anti-IgG for 30 minutes and washed again. The slides were examined with a Zeiss photomicroscope outfitted for transmission fluorescence microscopy using a primary KP490 (Zeiss) filter and a No. 30 barrier filter. Cells were considered to contain HSV antigens if they manifested a bright. apple-green nuclear and cytoplasmic fluorescence w-ith the immune reagents. EBectron Stdies MNL (107 cells/ml) were incubated at 37 C for 2 hours with medium containing 107 PFU of HSV or with ENMBS alone. After washing. cells were resuspended in fresh medium and cultured for 3 days at 37 C. Samples were then fixed in 0.05 NM sodium cacodylatebuffered (pH 7.2) 4% (- X glutaraldehyde for 1 hour at 4 C and postfixed in 0.1 IM sym-
collidine-buffered (pH 7.3) 15c (w v) OSO4. Following en bloc staining with 0.12 MI Veronal acetate-buffered (pH 4.7) 0.5% (w Xv) uranyl acetate, the cells were dehydiated in increasing concentrations of ethanol. Cell suspensions were centrifuged and the pellets were embedded in Epon 812. Thin and thick sections were cut on NIT-I Porter-Blum ultramicrotomes and stained with 7.3% uranyl magnesium acetate and 0.4%c lead citrate. Representative fields were examined with a Hitachi HU-1 1 E electron microscope at 73 k.
Results Growth of HSV in Normal MNL
Experiments were first conducted to confirm the reported observation that HSV' will replicate in PHA-stimulated MNL but not in normal unstimulated leukocvtes.l""2 When 106 MNL from normal immune and nonimmune donors were incubated for 3 davs with PHA and then exposed to HSV at a virus to cell ratio of 5, then 102 to 102 PFU of virus could be detected in these cultures immediately after treatment wvith anti-HSV' (Text-figure 1). The amount of virus in the MNL increased thereafter and attained maximum levels of 1065 to 1062 PFU w-ithin a davs after inoculation. In contrast, freshly isolated MNL did not replicate infectious virus, and the initial titers of 10'" to 102.6 declined to undetectable levels by Day 3. As shown, the immune status of the donor did not affect the replication of virus by the various MNL. thus, immune status determinations were considered unnecessary in subsequent experiments.
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TEXT-FIGLRE 1-The growth of HSV in MNL from normal donors. MNL (2 X 10ml) in EMBS w-ere either exposed to virus (10'-°PFU ml) or incubated with PHA (1 ALg ml) for 3 days and then
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infected. After treatment with anti-HSV, samples were incubated at 37 C for various intervals and assaved for virus content. Dotted lines indicate stimulated MN-L, solid lines indicate unstimulated MINL. = Donor 1 (immune): M = Donor 2 (immune). @ = Donor 3 (nonimmune):* Donor 4 (nonimmune).
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Abilty of HSV to Grow in Leukewic Cells
Days
We next determined if leukemic cells, like PHA-stimulated MNL, would support the growth of HSV (Text-figures 2 and 3). Only the cells from the 4 patients with HCL (Text-figure 2) showed evidence of virus growth, with maximum titers occurring within 1 to 7 days after inoculation. None of the HSV-exposed MNL from the 12 patients with CLL, LSCL, or ALL demonstrated a rise in virus titer during the interval studied (Text-figure 3). Fuoecet Antibody Studies of Infected MNL
To confirm the results obtained by virus assay, infected and uninfected NINL were examined for the presence of HSV antigens bv an indirect FA /p
TEXT-FIGURE 2-The growth of HSV in MNL from HCL patients. \NiNL (2 X 10' ml) in EMBS were exposed to virus (107 PFU ml), treated with antiHSV. cultivated for various intervals at 37 C, and then assayed for infectious virus. A= HCL-1; 0 = HCL-2: * = HCL-3;* HCL-4.
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TEXT-FIGURE :3-The ero\-th of HSV in \1NL from XLL A*. CLL 0 and LSCL U patients. N1N-L l2 X 1lr ml in E\MBS vvere exposed to virus (10-OPFU ml). treated mvith anti-HSV. cultivated for various intervals at 37 C. and then assax-ed for infectious virus. Values shown represent the averages for the M1NL from 9 CLL patients and from 2 LSCL patients. data for the ALL cells mvere oh-
Days
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patient.
technique using normal and immune rabbit serum samples. Infected. PHA-stimulated cells from the normal donors demonstrated fluorescence in the nucleus and cytoplasm only when treated with the immune serum. Similarly, infected HCL cells incubated 'with immune serum exhibited nuclear and cvtoplasmic fluorescence wvithin 1 day after virus inoculation (data not shown). One hundred to 300 of these infected MINL from each patient w,ere examined, and the percentage of cells containing HSV' antigens was determined. Fluorescence was found in 16, 14, 6. and 6% of the infected cells from patients HCL-1, HCL-2, HCL-3. and HCL-4, respectively. No fluorescence was observed when uninfected HCL cells were treated with the immune serum or wvhen infected HCL cells w-ere incubated w%ith nonimmune serum. Ectro Microscopic Studies of Infected MNL
V'irus growth was also assessed by an ultrastructural study of the infected cells. Cultures of PHA-stimulated MNL and HCL cells wvere examined 3 days after incubation with HSV' or media. HSV' capsid structures wvere seen within the nucleus of 6 to 8% of the PHA-stimulated and HCL MNL after exposure to virus (Table 1 and Figure 1). No virus particles were noted in 100 uninfected HCL cells cultivated for .3 days (Table 1 ). Infect Stdies
A possible explanation for the observed differences in the production of virus by various MNL might lie in the ability of HSV' to adsorb to these different cell types. Neither HCL nor CLL cells adsorbed significant amounts of virus (less than 0.3 Iog10) wvhen assessed by the ability of the cells to deplete the supernatant of virus. This impression was confirmed using a more sensitive virus adsorption assay. The method consisted of quantitating the uptake of 3H-labeled virus by HCL cells as compared with normal unstimulated MNL (control). Of the 200,000 cpm of radio-
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Table 1-Intranuclear Herpesvirus Capsid Structures in Infected HCL and PHA-stimulated Normal MNL
Source of MNL
Treatment of MNL'
HCL-1
Infected
HCL-1 Normal
Uninfected PHA-treated and infected
No. of MNL examined
Percent of cells with intranuclear virus particles
200 200 100 150
8 6 0 6
Cells were incubated with virus or media, cultivated for 3 days, and processed for electron microscopic study.
labeled virus incubated with the cells, approximately 1000 cpm was adsorbed by either HCL or control cells. This indicated that only 0.5% of the inoculum wvas adsorbed by the leukocvtes, and there appeared to be no difference between MNL which could support the replication of virus (HCL cells) and those which showed no evidence of HSV growth (normal MNL). Further experiments investigated the ability of virus to penetrate various MINL. The number of PFU of intracellular virus within the cells after exposure to virus, treatment with anti-HSV, wvashing, and sonication is show-n in Table 2. Considerable variation existed in the amount of virus found wvithin the normal NINL, wvith an average of 84 PFU being released from 106 cells. When MNL from these same patients were exposed to PHA and then infected in an identical fashion, the amount of virus w1ithin these cells (107 PFU) was not substantially different from that of unstimulated NINL. Slightly higher levels of intracellular virus (130 to 455 PFU) w%vere found in HCL cells. These differences in the amounts of virus within the normal, PHA-stimulated, and HCL cells, however, could not account for the observed differences in virus growth (Text-figures 1 and 2). Table 2-Assessment of Virus PeneLration and Production of HSV by Normal and HCL MNL
Source of MNL Normal Normal HCL-1 HCL-4
Treatment of MNL'
PFU of intracellulart virus per 10 cells
Infectious centers per 10. cellst
Infected PHA-treated and infected Infected Infected
84 ± 21.5§ 107 ± 67§ 455 130
2.8 ± 0.6§ 1029 ± 106§ 1043 240
' Cells were incubated with HSV, washed, treated with anti-HSV, washed again, and resuspended in medium. t The concentration of intracellular virus was determined 3 hours after infection, following freeze-thawing and sonicating the MNL. t Ten-fold dilutions of infected cells were layered onto PRK monolayers and overlaid with methocel. Infectious centers were assessed at 3 days. § Average values obtained from 4 donors ± SEM.
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The ability of the cell to uncoat, replicate, and release infectious virus was cumulatively assessed by infectious center assays, in which the number of MNL producing or releasing virus is determined. Cells from 4 normal donors, with or without PHA-stimulation, and from 2 HCL patients were examined. The number of infectious centers produced by 106 MNL is also shown in Table 2. Few infectious centers (2.8 ± 0.6) were produced by normal unstimulated MNL; however, prior PHA treatment of cells from the same donors resulted in a marked increase in the number of MNL capable of producing infectious virus (1029 ± 106 infectious centers). A similar value (1043) was obtained following infection of MNL from a patient with HCL whose MNL fraction consisted of 99.5% leukemic cells; MNL from a second HCL patient with 50% leukemic cells produced fewer (240) infectious centers.
Productive infection of normal MNL by HSV in vitro does not occur unless the cells are mitogen-stimulated and the immune status of the donor does not affect the ability of the leukocyte to replicate virus (Textfigure 1).10-u The experiments reported here demonstrate that, with the exception of HCL cells, freshly isolated leukemic MNL from patients with CLL, LSCL, or ALL are also incapable of replicating HSV. Whether these leukemic cells could be induced to support the growth of HSV by mitogen stimulation is not known, and technical difficulties in obtaining purified populations of leukemic cells would hamper the interpretation of such experiments. The fact that freshly isolated MNL do not replicate virus would be evidence against the possibility of HSV disseminating within the body via virus-replicating leukemic cells. Although this method of virus spread might occur in HCL patients, there are few data to suggest that these individuals have an increased susceptibility to disseminated herpetic disease. The propensity of patients with lymphosarcoma,'6 ALL,1 and CLL 15 to develop extensive HSV infections must, therefore, be attributed to therapy, the immune deficit that accompanies these diseases, or other unknown factors. MNL from HCL patients, regardless of the patient's previous treatment or clinical course, consistently demonstrated replication of HSV (Textfigure 2). Infection of these cells was confirmed by FA and ultrastructural studies (Figure 1). Our data indicate that the hairy cells, rather than nonleukemic cells in the MNL fractions, replicated the virus. This conclusion was based on studies done with leukocytes isolated from the blood of patient HCL-1, whose MNL fraction consisted of 99.5% hairy cells. After infection, 6 to 8% of his leukemic cells showed ultrastructural evidence of virus produc-
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tion (Table 1), and a slightly higher proportion (14%) of these leukocytes contained HSV antigens, as shown by FA studies. Although one laboratory has reported finding herpesvirus nucleocapsid structures in hairy cells after in vitro cultivation,31 we found no evidence of herpesvirus on electron microscopy or of HSV antigens on FA examination in uninfected cells after 3 days of culture. Further experiments attempted to explain why HCL and PHA-stimulated cells replicated HSV while normal and other leukemic cells did not. Differences in the ability of the virus to adsorb, penetrate, uncoat, and replicate within the various MNL were considered. Our experiments suggested that adsorption of HSV occurred equally well in HCL cells and normal unstimulated MNL. Furthermore, quantitation of intracellular virus during the early stages of infection demonstrated no significant differences in the ability of the virus to penetrate HCL cells in comparison to PHA-stimulated or normal MNL (Table 2). Productive infection of MNL by HSV thus appeared to depend on the ability of the leukocyte to support a later stage of infection, either uncoating or replication. This assertion was substantiated by the infectious center data shown in Table 2. Although virus was capable of gaining entry into the unstimulated normal MNL, this leukocyte inactivated the virus, and few infectious centers were formed. A productive infection, rather than inactivation of the virus, occurred within PHA-stimulated or HCL cells, and the number of infectious centers produced was much greater. Quantitative consideration of the data indicated that only a small fraction of the HCL cells participated in virus production. Similar results were found with PHA-stimulated normal MNL. When 10 cells were exposed to an excess of virus, only 1000 (0.1%) produced infectious virus (Table 2). However, FA examination of these cells demonstrated HSV antigens in 6 to 16%, and 6 to 8% of the cells showed ultrastructural evidence of virus replication (Table 1). These data suggest that the majority of the HCL cells do not become infected when exposed to HSV; of those that do become infected, only a small percentage produce infectious virus. The exact percentage of abortively infected cells is uncertain since cell-to-cell spread of virus may have increased the number of infected cells measured by electron microscopy or FA methods. A point that remains unclear is why hairy cells, in contrast to other leukemic cells, were capable of supporting the growth of HSV. One possibility would be that the ability to grow HSV is influenced by the series (monocytic, B-lymphocytic, or T-lymphocytic) from which the malignant cell arose. At present, a dispute exists concerning the clonal origin of the malignant cell in HCL. Some investigators have presented evidence
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for a monocytic origin,32 whereas others have data supporting a B-l-mphocytic origin of this leukocvte.3-35 In the murine system, HSV has been shown to be incapable of productively infecting mitogen-stimulated T lymphocytes." Murine monocvtes and blast-transformed B lymphocytes. how ever, are able to replicate infectious virus. 3 3 Theoretically, either monocvtes or B lymphocytes could have the potential for replicating HSV. Howvever, if HCL cells are of the B-lvmphocvtic series, as most data now suggest, then the type of cell alone could not account for the ability of the leukocvte to grow virus, since replication of HSV was not observed in CLL or LSCL cells which had B-cell characteristics. Other factors, perhaps unique to HCL cells, such as metabolic activity or degree of differentiation, must also influence the ability of leukemic cells to replicate HSV. References 1. Berg RB: Multiplication of Echo 9 virus in suspensions of human leukocytes. Proc Soc Exp Biol Med 108:742-744. 1961 2. Gresser I. Chany C: Multiplication of poliovirus type I in preparations of human leukocytes and its inhibition by interferon. J Immunol 92:889-893. 1964 .3. Edelman R, Wheelock EF: V'esicular stomatitis virus replication in human leukocvte cultures: Enhancement by ph%tohemagglutinin. Science 154:1053-1055. 1966 4. Edelman R, Wheelock EF: Specific role of each human leukocyte type in viral infections. I. Monocvte as host cell for vesicular stomatitis virus replication in vitro. J V'irol 1:1139-1149, 1967 3. Miller G, Enders JF: Vaccinia virus replication and cytopathic effect in cultures of phv-tohemagglutinin-treated human peripheral blood leukocvtes. J Virol 2:787-792. 1968 6. Wheelock EF. Edelman R: Specific role of each human leukocvte type in viral infections. III. 17D yellow fever virus replication and interferon production in homogeneous leukocv-te cultures treated w-ith phvtohemagglutinin. J Immunol I a3:429-436, 1969 7. Lambriex NM. van der Veen J: Comparison of replication of adenovirus type 2 and type 4 in human lymphoc\-te cultures. Infect Immun 14:618-622. 1976 8. Edelman R. Wheelock EF: Specific role of each human leukocyte type in viral infections. II. Phytohemagglutinin-treated lymphocytes as host cells for vesicular stomatitis virus replication in citro. J Virol 2:440-448 1968 9. W%7illems FTC, Melnick JL, Rawls WVE: Replication of poliovirus in phvtohemagglutinin-stimulated human lymphocy%tes. J Virol :3:431-457, 1969 10. N.ahmias AJ, Kibrick S, Rosan RC: Viral leukocvte interrelationships. I. Multiplication of a DNA virus-herpes simplex-in human leukocv-te cultures. J Immunol 93:69-74, 1964 11. Bouroncle BA, Clausen KP. Damer EM: Replication of herpes simplex virus in cultures of phy-tohemagglutinin-stimulated human lymphocytes. J Natl Cancer Inst 44:1065-1078, 1970 12. Kleinman LF. Kibrick S, Ennis F. Polgar P: Herpes simplex virus replication in human lymphocyte cultures stimulated with phytomitogens and anti-lymphocyte globulin. Proc Soc Exp Biol Med 141:1095-1099. 1972 13. Henle VN7, Henle G. zurHausen H: Effect of herpes simplex v-irus on cultured Burkitt tumor cells and its failure to influence the Epstein-Barr virus carrier state. Cancer Res 29:489-494, 1969
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14. Floyd R, Glasser R. Vonka V, Benvesh-Melnick MI: Studies on the growth of herpes simplex virus in lvsmphoblastoid cells. Acta V'irol (Praha) 15:133-142, 1971 15. Gerber A, Sauter C. Lindenmann J: Fowl plaque virus adapted to human epithelial tumor cells and human mveloblasts in vitro. II. Replication in human leukemic mveloblast culture. Arch Ges Virusforsch 40:255-264, 1973 16. Muller SA, Herrrnann EC Jr. Winkelmann RK: Herpes simplex infections in hematologic malignancies. Am J Med 52:10'2-114. 1972 17. Cappel R, Klastersky J: Herpetic meningitis (type 1) in a case of acute leukemia. Arch Neurol 28:415-416, 1973 18. Habel K: Virus neutralization test. Fundamental Techniques in Virology. Edited by K Habel, NP Salzman. New York. Academic Press. Inc.. 1969, pp 291-293 19. Cohen HJ: Human lymphocyte surface immunoglobulin capping: Normal characteristics and anomalous behavior of chronic lymphocy-tic leukemic lymphocytes. J Clin Invest 55:84-93, 1975 20. Jarvis SC, Snyderman R. Cohen HJ: Human lymphocyte motility: Normal characteristics and anomalous behavior of chronic lymphocvtic leukemia cells. Blood 48:7 1 -729, 1976 21. Aisenberg AC. Wilkes B: Lymphosarcoma cell leukemia: The contribution of cell surface study to diagnosis. Blood 48:707-715. 1976 22. Catovskv D, Pettit JE, Galton DAG. Spiers ASD. Harrison CV: Leukemic reticuloendotheliosis ('Hairy' Cell Leukaemia). A distinct clinico-pathological entity. Br J Haematol 26:9-27. 1974 23. Hamper B, Notkins AL, Mage NIt, Keehn MA: Heterogeneity in the properties of 7-S and 19S rabbit-neutralizing antibodies to herpes simplex virus. J Immunol 100:386-593. 1968 24. Ship II. Ashe WK, Scherp HW: Recurrent "fever blister" and "canker sore." Tests for herpes simplex and other viruses with mammalian cell cultures. Arch Oral Biol 3:117-124. 1961 23. Shinkai K: Plaque morphology of herpes simplex virus in various cells under liquid overlay as a marker for its type differentiation. Jpn J Microbiol 19:459-462. 1973 26. Ashe WVK. Scherp HN: Antigenic analysis of herpes simplex virus by neutralization kinetics. J Immunol 91:658-6653 1963 27. Notkins AL. Rosenthal J. Johnson B: Rate-zonal centrifugation of herpes simplex virus-antibody complexes. Virology 4.3:321-325. 1971 28. Bvurum A: Isolation of mononuclear cells and granulocvtes from human blood. Scand J Clin Lab Invest 21 (Suppl 97):77-89. 1968 29. Theofilopoulos AN. Brandt WVE. Russell PK. Dixon FT: Replication of dengue-2 virus in cultured human lymphoblastoid cells and subpopulations of human peripheral leukocvtes. J Immunol 117:953-961, 1976 .30. Porath J, Aspberg K. Drevin H, Axen R: Preparation of cyanogen bromide activated agarose gels. J Chromatogr 86:33-36. 1973 31. Sinkovics JG. Gy6rkey F. Gvorkev- P: Leukemic reticuloendotheliosis and "hairy cells" 293:1016. 1976 32. Scheinberg NI. Brenner Al. Sullivan AL. Cathcart ES. Katavama I: The heterogeneity of leukemic reticuloendotheliosis. "hairy cell leukemia." Evidence for its monocytic origin. Cancer 37:1302-1307d 1976 .33 Deegan NIJ, Cossman J. Chosney BT. Schnitzer B: Hairy cell leukemia. An immunologic and ultrastructural study. Cancer '38:1932-1961. 1976 34. Utsinger PD. Yount WJ, Fuller CR. Logue NIJ, Orringer EP: Hairy cell leukemia: B-lymphocyte and phagocytic properties. Blood 49:19-27. 1977 .35. Haak HL. deNMan JCH. Hijmans WV, Knapp XV. Speck B: Further evidence for the lvmphocytic nature of leukaemic reticuloendotheliosis (hairy cell leukaemia). Br J Haematol 27:31-.38, 1974
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36. Kirchner H, Hirt HM, Kleinicke C, Munk K: Replication of herpes simplex virus in mouse spleen cell cultures stimulated by lipopolysaccharide. J Immunol 117:1753-1756, 1976 37. Johnson RT: The pathogenesis of herpes virus encephalitis. II. A cellular basis for the development of resistance with age. J Exp Med 120:359-374, 1964 38. duBuy H: Effect of silica on virus infections in mice and mouse tissue culture. Infect Immun 11:996-1002, 1975
Acowledgmnts Excellent technical assistance was provided by Mrs. Sylvia LeGoff and Mr. Thomas Lassater. We also wish to thank Mrs. Frances Slocum for manuscript preparation.
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IA-Electron IIAmicrograph of an HSV-infected MNL from a patient with hairy cell leukemiaa after 3 days of)f culture. cufture. Fqu Fe" At the periphery of the cell, microvillous structures can be seen. The contains numerous imerous fat
cytoplasm osmiophilic 3philic per i droplets, matin is clumped mitochondria, and dilated endoplasmic reticulum. Within the nucleus, chromatin )ed at the droplet swollen nuclear mi membrane, and several clusters of nucleocapsid structures can be seen adjacent to this his aggregated Ichromatin (arrows). Je the cell an enveloped (arri (arrom B-Two herpesvirus capsid structures can be seen in the nucleoplasm and outside -nveloped virus particle is present. (Uranyl magnesium acetate and lead citrate, A x 16,800; B x 105,000) pparti '000)
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[End of Article]
The ability of leukemic leukocytes to support the replication of herpes simplex virus (HSV) was studied. Mononuclear leukocytes (MNL) from the peripheral blood of patients with a variety of lymphoid leukemias were isolated on Ficoll-Hypaque gradients and infected with HSV at a multiplicity of infection of 5 to 10. No virus growth was detected in cells from patients with chronic lymphocytic leukemia (9), acute lymphocytic leukemia (1), or lymphosarcoma cell leukemia (2). HSV replication did occur in hairy cell leukemic MNL from all of 4 patients studied. Maximal titers of 10' to 10'" PFU/ml occurred 1 to 7 days after incubation. By electron microscopy, herpesvirus particles were seen in the nuclei of these infected cells after 3 days of culture, but none was seen in the cells not exposed to virus. Fluorescent antibody mition confirmed the presence of HSV antigens in the nuclei of infected hairy cells. No difference in the adsorption or penetration of the virus was found with the various MNL studied. Productive infection of the cells thus appeared to depend on the ability of the leukocyte to support a later stage of infection, either uncoating or replication of the virus. (Am J Pathol 90:187-200, 1978)
A VARIE OF VIRUSES have been shown to be capable of replicating in human mononuclear leukocytes (MNL).1-7 The majority of these agents are able to productively infect freshly isolated MNL, and treatment of the cells with mitogen prior to infection markedly increases the amount of virus produced.3'6' Herpes simplex virus (HSV) is an exception, in that this agent will not productively infect freshly isolated MNL but will grow only in mitogen-stimulated leukocytes 1012 or continuous lymphoblastoid cell lines. lo Few investigators have studied the ability of leukemic leukocvtes to replicate virus. Gerber et al1 found that an avian myxovirus could grow in primary leukocyte cultures from patients with acute myelogenous leukemia, but little is known conceming the ability of human viruses to grow in freshly isolated leukemic lymphocytes. Since patients with leukemia have a propensity toward developing extensive and sometimes disseminated HSV infections,""'7 we reasoned that replication of this virus in From the Department of Pathology, Duke University Medical Center, and Department of Medicine, Division of Hematology, Veterans Administration Hospital, Durham, North Carolina. Supported in part by Grant CA 05634-16 from the National Cancer Institute and Grant DE 04609 from the National Institute of Dental Research, National Institutes of Health. Accepted for publication August 23, 1977. Presented in part at the Sixtv-first Annual Meeting of the Federation of American Societies for Experimental Biology, April 7, 1977, Chicago, Illinois. Address reprint requests to Dr. Charles A. Daniels, Department of Pathology, Duke Universitv 'Medical Center, Durham. NC 27i10.
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malignant leukocytes might play a role in the spread of infection. In.an effort to explore this hypothesis, we determined whether the lymphoid cells from the peripheral blood of patients with hairy cell (HCL), chronic lymphocytic (CLL), acute lymphocytic (ALL), and lymphosarcoma cell (LSCL) leukemia would support the replication of HSV. Materials and NWNMethodsPamb
Sixteen patients with malignant lymphoproliferative diseases and 4 normal subjects were studied, all of whom consented to have blood drawn for study. The normal subjects were classified as immune or nonimmune on the basis of the presence of anti-HSV antibodies in their heat-inactivated serum samples.u Nonimmune donors had no detectable neutralizing activity in their serum samples when they were tested at a 1:10 dilution, whereas immune subjects' serum samples had titers of greater than 1: 100. Nine patients, aged 56 to 74, had peripheral leukocyte abnormalities characteristic of CLL, including surface membrane immunoglobulin.ls. Four of these individuals were untreated; the remainder were receiving cytotoxic chemotherapy. Two patients with a history of nonHodgkin lymphoma had elevated peripheral blood leukocyte counts with a predominance of lymphoid cells characteristic of those seen in LSCL, including dense surface membrane immunoglobulin.2' Neither had received chemotherapy within 3 weeks of study. In addition, a 26-year-old patient was studied who had relapsed ALL with lymphoblasts comprising more than 95% of his peripheral blood leukocytes. Cells were also obtained from 4 patients with HCL, none of whom had received chemotherapy. Each patient initially presented with splenomegaly, anemia, thrombocvtopenia, and diffuse infiltration of the bone marrow by abnormal lymphoid cells. All 4 had peripheral blood mononuclear cells characteristic of HCL, including long filamentous projections by phase microscopy and the presence of intracellular tartrate-resistant acid phosphatase.22 Patient HCL-1, with a peripheral leukocyte count of 40,000 per cu mm and 99.5% HCL cells, had previously undergone splenectomy without demonstrable benefit. Patients HCL-2 and HCL-3 had been treated by splenectomy with partial resolution of anemia and thrombocvtopenia. Patient HCL-4 was untreated.
Vv1 md Antswwm HSV, strain CHR-HSV-3,23 was isolated from a patient with recurrent herpes labialis and identified as Type I HSV by virus neutralization tests using immune rabbit serum'4 and by plaque morphology on chick embryo fibroblasts.2 The virus, grown in human embryonic foreskin fibroblasts (Flow Laboratories, Bethesda, Md.), was assayed as plaqueforming units (PFU) on primary rabbit kidney (PRK) cells using an antibody overlay 0 and had a titer of 10"' to 10Z' PFU/ml. sH-labeled HSV was prepared by addition of 'Hthymidine (New England Nuclear, Boston, Mass.) to infected PRK cells according to the method of Notkins et al.'7 Human HSV immune serum samples were obtained from patients with recurrent herpetic infections. The serum samples, after heat inactivation at 56 C for 30 minutes, had neutralization titers of 1: 256 or greater as determined by the plaque reduction method."' Rabbit serum samples were obtained from adult New Zealand white rabbits either prior to immunization (normal rabbit serum samples) or 4 weeks after injection with PRK-grown HSV (immune serum samples)." HSV neutralization titers of normal rabbit serum samples were less than 1: 10, and those of immune serum samples were 1: 10,000.
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Isoation Iof Human Monnl Lesw cytgs
Ten to thirtv milliliters of venous blood was collected in heparinized (10 units/ml) svringes (Upjohn Co., Kalamazoo, Mich.) from each leukemic patient and each healthv donor. MNL were isolated from the whole blood samples by centrifugation on FicollHypaque gradients. The cells were washed twice in Eagle's minimal essential medium supplemented with 10% (v/v) fetal bovine serum, 50 jig/ml streptomvcin, and 100 units/ml penicillin G (EMBS),* and the concentration was adjusted to 2 X 10' cells/ml. The MNL from the leukemic patients were exposed to virus within 24 hours of isolation. When indicated in the text, normal MNL were either incubated with virus within 1 day of separation or exposed to phytohemagglutinin (PHA) and then infected with HSV. These normal cells were stimulated by incubating the MNL (2 X 10' cells/ml) with an equal volume of EMBS containing 2 pg/ml PHA (Wellcome Laboratories, Beckenham, England) for 3 days at 37 C and resuspended in fresh medium prior to infection. inftion of MNL
An equal volume of EMBS containing HSV (107- to 107 PFU/ml) was added to an MNL suspension (10"' cells/ml) (multiplicitv of infection of 5 to 10), and samples were incubated for 2 hours at 37 C. After washing twice, cells were treated for 60 minutes with human anti-HSV serum (1:4 dilution) to neutralize the unpenetrated virus and to synchronize the infection. MNL were washed again, dispensed into 1-mi portions containing 10' cells, and cultured at 37 C in a CO, incubator for intervals up to 7 days. At the times indicated, samples were removed and frozen at -70 C.
hb 4mSAssms MNL cultures were thawed and sonicated at 50 watts for 30 seconds with a Sonifier Cell Disrupter (Model W135, Heat Systems Ultrasonics, Inc., Plainview, NY). Cell debris was removed by centrifugation (300 X g for 10 minutes), and the supernatants wer-e assayed for infectivity. When virus replication in the leukocytes was demonstrated, i.e., an increase in virus titer of 0.3 log1, PFU/ml above the Day 0 value, the identity of the agent as Type I HSV was established by virus neutralization tests using immune rabbit serum 24 and by plaque morphology on chick embryo fibroblasts.2u In addition to the above, the number of infected cells was quantitated by an infectious center assay.5 Following treatment with antiviral serum and washing, the HSV-exposed MNL were adjusted to 10' cells/ml. One-tenth milliliter of serial 10-fold dilutions of each sample were plated in duplicate onto PRK monolayers. Each dish was overlaid with 4 ml of EMBS containing 2% (w/v) methvl cellulose (Methocel), and the plaques were counted after 3 days. po i Aup VAm 'H-labeled virus (200,000 cpm/ml) was incubated for 2 hours at 4 C with an equal volume of medium containing 2 X 10' MNL. Cells were washed three times, and the radioactivitv associated with the MNL was determined using a Packard Tricarb Scintillation Spectrophotometer (Packard Instruments, Chicago, Ill.). Adsorption studies were also performed by assessing the amount of virus depleted from the medium by the MNL.' HSV (2 X 10' PFU) was incubated with a suspension of 2 X 10' MNL or with medium alone (control). After 2 hours at 4 C, cells were centrifuged, and residual virus in the supematants was assaved for infectivity and compared with the control. * Unless otherwise stated, all washing procedures and dilutions of cells. virus. and antisenrms sere done in E%1BS.
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Floecn Antbody Stdies Infected and uninfected MNL were examined for HSV antigens by an indirect fluores-
cent antibody (FA) method. Rabbit serum samples. obtained as described above. were absorbed with uninfected human MNL and diluted 1: 10 in phosphate-buffered (pH 7.3) sodium chloride solution (PBS) containing 4%5 (w v) bovine serum albumin (BSA). Goat antirabbit IgG (heavy chain specific) conjugated with fluorescein isothiocyanate (FITC) (Cappel Laboratories, Downington, Pa.) was absorbed with human 5-globulin conjugated to Sepharose 4B ° and diluted 1 :2 in PBS-BSA. After 0. 1. 3. and 3 days of culture. cells were fixed to glass slides by spinning at 260 X g for 10 minutes on a cytocentrifuge (Shandon Southern Instruments, Sewickley. Pa.). The air-dried slides w-ere washed in PBS. fixed in acetone for 5 minutes at 25 C, and washed again in PBS. Each slide was then treated for 30 minutes with either normal or immune rabbit serum. Follow ing w-ashing in PBS. each slide was reacted with the FITC-conjugated anti-IgG for 30 minutes and washed again. The slides were examined with a Zeiss photomicroscope outfitted for transmission fluorescence microscopy using a primary KP490 (Zeiss) filter and a No. 30 barrier filter. Cells were considered to contain HSV antigens if they manifested a bright. apple-green nuclear and cytoplasmic fluorescence w-ith the immune reagents. EBectron Stdies MNL (107 cells/ml) were incubated at 37 C for 2 hours with medium containing 107 PFU of HSV or with ENMBS alone. After washing. cells were resuspended in fresh medium and cultured for 3 days at 37 C. Samples were then fixed in 0.05 NM sodium cacodylatebuffered (pH 7.2) 4% (- X glutaraldehyde for 1 hour at 4 C and postfixed in 0.1 IM sym-
collidine-buffered (pH 7.3) 15c (w v) OSO4. Following en bloc staining with 0.12 MI Veronal acetate-buffered (pH 4.7) 0.5% (w Xv) uranyl acetate, the cells were dehydiated in increasing concentrations of ethanol. Cell suspensions were centrifuged and the pellets were embedded in Epon 812. Thin and thick sections were cut on NIT-I Porter-Blum ultramicrotomes and stained with 7.3% uranyl magnesium acetate and 0.4%c lead citrate. Representative fields were examined with a Hitachi HU-1 1 E electron microscope at 73 k.
Results Growth of HSV in Normal MNL
Experiments were first conducted to confirm the reported observation that HSV' will replicate in PHA-stimulated MNL but not in normal unstimulated leukocvtes.l""2 When 106 MNL from normal immune and nonimmune donors were incubated for 3 davs with PHA and then exposed to HSV at a virus to cell ratio of 5, then 102 to 102 PFU of virus could be detected in these cultures immediately after treatment wvith anti-HSV' (Text-figure 1). The amount of virus in the MNL increased thereafter and attained maximum levels of 1065 to 1062 PFU w-ithin a davs after inoculation. In contrast, freshly isolated MNL did not replicate infectious virus, and the initial titers of 10'" to 102.6 declined to undetectable levels by Day 3. As shown, the immune status of the donor did not affect the replication of virus by the various MNL. thus, immune status determinations were considered unnecessary in subsequent experiments.
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HSV REPLICATION IN HAIRY CELL LEUKEMIC LEUKOCYTES
TEXT-FIGLRE 1-The growth of HSV in MNL from normal donors. MNL (2 X 10ml) in EMBS w-ere either exposed to virus (10'-°PFU ml) or incubated with PHA (1 ALg ml) for 3 days and then
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1-
0 I-
infected. After treatment with anti-HSV, samples were incubated at 37 C for various intervals and assaved for virus content. Dotted lines indicate stimulated MN-L, solid lines indicate unstimulated MINL. = Donor 1 (immune): M = Donor 2 (immune). @ = Donor 3 (nonimmune):* Donor 4 (nonimmune).
>0
Qj -
Abilty of HSV to Grow in Leukewic Cells
Days
We next determined if leukemic cells, like PHA-stimulated MNL, would support the growth of HSV (Text-figures 2 and 3). Only the cells from the 4 patients with HCL (Text-figure 2) showed evidence of virus growth, with maximum titers occurring within 1 to 7 days after inoculation. None of the HSV-exposed MNL from the 12 patients with CLL, LSCL, or ALL demonstrated a rise in virus titer during the interval studied (Text-figure 3). Fuoecet Antibody Studies of Infected MNL
To confirm the results obtained by virus assay, infected and uninfected NINL were examined for the presence of HSV antigens bv an indirect FA /p
TEXT-FIGURE 2-The growth of HSV in MNL from HCL patients. \NiNL (2 X 10' ml) in EMBS were exposed to virus (107 PFU ml), treated with antiHSV. cultivated for various intervals at 37 C, and then assayed for infectious virus. A= HCL-1; 0 = HCL-2: * = HCL-3;* HCL-4.
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TEXT-FIGURE :3-The ero\-th of HSV in \1NL from XLL A*. CLL 0 and LSCL U patients. N1N-L l2 X 1lr ml in E\MBS vvere exposed to virus (10-OPFU ml). treated mvith anti-HSV. cultivated for various intervals at 37 C. and then assax-ed for infectious virus. Values shown represent the averages for the M1NL from 9 CLL patients and from 2 LSCL patients. data for the ALL cells mvere oh-
Days
tained from an individual
patient.
technique using normal and immune rabbit serum samples. Infected. PHA-stimulated cells from the normal donors demonstrated fluorescence in the nucleus and cytoplasm only when treated with the immune serum. Similarly, infected HCL cells incubated 'with immune serum exhibited nuclear and cvtoplasmic fluorescence wvithin 1 day after virus inoculation (data not shown). One hundred to 300 of these infected MINL from each patient w,ere examined, and the percentage of cells containing HSV' antigens was determined. Fluorescence was found in 16, 14, 6. and 6% of the infected cells from patients HCL-1, HCL-2, HCL-3. and HCL-4, respectively. No fluorescence was observed when uninfected HCL cells were treated with the immune serum or wvhen infected HCL cells w-ere incubated w%ith nonimmune serum. Ectro Microscopic Studies of Infected MNL
V'irus growth was also assessed by an ultrastructural study of the infected cells. Cultures of PHA-stimulated MNL and HCL cells wvere examined 3 days after incubation with HSV' or media. HSV' capsid structures wvere seen within the nucleus of 6 to 8% of the PHA-stimulated and HCL MNL after exposure to virus (Table 1 and Figure 1). No virus particles were noted in 100 uninfected HCL cells cultivated for .3 days (Table 1 ). Infect Stdies
A possible explanation for the observed differences in the production of virus by various MNL might lie in the ability of HSV' to adsorb to these different cell types. Neither HCL nor CLL cells adsorbed significant amounts of virus (less than 0.3 Iog10) wvhen assessed by the ability of the cells to deplete the supernatant of virus. This impression was confirmed using a more sensitive virus adsorption assay. The method consisted of quantitating the uptake of 3H-labeled virus by HCL cells as compared with normal unstimulated MNL (control). Of the 200,000 cpm of radio-
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Table 1-Intranuclear Herpesvirus Capsid Structures in Infected HCL and PHA-stimulated Normal MNL
Source of MNL
Treatment of MNL'
HCL-1
Infected
HCL-1 Normal
Uninfected PHA-treated and infected
No. of MNL examined
Percent of cells with intranuclear virus particles
200 200 100 150
8 6 0 6
Cells were incubated with virus or media, cultivated for 3 days, and processed for electron microscopic study.
labeled virus incubated with the cells, approximately 1000 cpm was adsorbed by either HCL or control cells. This indicated that only 0.5% of the inoculum wvas adsorbed by the leukocvtes, and there appeared to be no difference between MNL which could support the replication of virus (HCL cells) and those which showed no evidence of HSV growth (normal MNL). Further experiments investigated the ability of virus to penetrate various MINL. The number of PFU of intracellular virus within the cells after exposure to virus, treatment with anti-HSV, wvashing, and sonication is show-n in Table 2. Considerable variation existed in the amount of virus found wvithin the normal NINL, wvith an average of 84 PFU being released from 106 cells. When MNL from these same patients were exposed to PHA and then infected in an identical fashion, the amount of virus w1ithin these cells (107 PFU) was not substantially different from that of unstimulated NINL. Slightly higher levels of intracellular virus (130 to 455 PFU) w%vere found in HCL cells. These differences in the amounts of virus within the normal, PHA-stimulated, and HCL cells, however, could not account for the observed differences in virus growth (Text-figures 1 and 2). Table 2-Assessment of Virus PeneLration and Production of HSV by Normal and HCL MNL
Source of MNL Normal Normal HCL-1 HCL-4
Treatment of MNL'
PFU of intracellulart virus per 10 cells
Infectious centers per 10. cellst
Infected PHA-treated and infected Infected Infected
84 ± 21.5§ 107 ± 67§ 455 130
2.8 ± 0.6§ 1029 ± 106§ 1043 240
' Cells were incubated with HSV, washed, treated with anti-HSV, washed again, and resuspended in medium. t The concentration of intracellular virus was determined 3 hours after infection, following freeze-thawing and sonicating the MNL. t Ten-fold dilutions of infected cells were layered onto PRK monolayers and overlaid with methocel. Infectious centers were assessed at 3 days. § Average values obtained from 4 donors ± SEM.
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The ability of the cell to uncoat, replicate, and release infectious virus was cumulatively assessed by infectious center assays, in which the number of MNL producing or releasing virus is determined. Cells from 4 normal donors, with or without PHA-stimulation, and from 2 HCL patients were examined. The number of infectious centers produced by 106 MNL is also shown in Table 2. Few infectious centers (2.8 ± 0.6) were produced by normal unstimulated MNL; however, prior PHA treatment of cells from the same donors resulted in a marked increase in the number of MNL capable of producing infectious virus (1029 ± 106 infectious centers). A similar value (1043) was obtained following infection of MNL from a patient with HCL whose MNL fraction consisted of 99.5% leukemic cells; MNL from a second HCL patient with 50% leukemic cells produced fewer (240) infectious centers.
Productive infection of normal MNL by HSV in vitro does not occur unless the cells are mitogen-stimulated and the immune status of the donor does not affect the ability of the leukocyte to replicate virus (Textfigure 1).10-u The experiments reported here demonstrate that, with the exception of HCL cells, freshly isolated leukemic MNL from patients with CLL, LSCL, or ALL are also incapable of replicating HSV. Whether these leukemic cells could be induced to support the growth of HSV by mitogen stimulation is not known, and technical difficulties in obtaining purified populations of leukemic cells would hamper the interpretation of such experiments. The fact that freshly isolated MNL do not replicate virus would be evidence against the possibility of HSV disseminating within the body via virus-replicating leukemic cells. Although this method of virus spread might occur in HCL patients, there are few data to suggest that these individuals have an increased susceptibility to disseminated herpetic disease. The propensity of patients with lymphosarcoma,'6 ALL,1 and CLL 15 to develop extensive HSV infections must, therefore, be attributed to therapy, the immune deficit that accompanies these diseases, or other unknown factors. MNL from HCL patients, regardless of the patient's previous treatment or clinical course, consistently demonstrated replication of HSV (Textfigure 2). Infection of these cells was confirmed by FA and ultrastructural studies (Figure 1). Our data indicate that the hairy cells, rather than nonleukemic cells in the MNL fractions, replicated the virus. This conclusion was based on studies done with leukocytes isolated from the blood of patient HCL-1, whose MNL fraction consisted of 99.5% hairy cells. After infection, 6 to 8% of his leukemic cells showed ultrastructural evidence of virus produc-
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tion (Table 1), and a slightly higher proportion (14%) of these leukocytes contained HSV antigens, as shown by FA studies. Although one laboratory has reported finding herpesvirus nucleocapsid structures in hairy cells after in vitro cultivation,31 we found no evidence of herpesvirus on electron microscopy or of HSV antigens on FA examination in uninfected cells after 3 days of culture. Further experiments attempted to explain why HCL and PHA-stimulated cells replicated HSV while normal and other leukemic cells did not. Differences in the ability of the virus to adsorb, penetrate, uncoat, and replicate within the various MNL were considered. Our experiments suggested that adsorption of HSV occurred equally well in HCL cells and normal unstimulated MNL. Furthermore, quantitation of intracellular virus during the early stages of infection demonstrated no significant differences in the ability of the virus to penetrate HCL cells in comparison to PHA-stimulated or normal MNL (Table 2). Productive infection of MNL by HSV thus appeared to depend on the ability of the leukocyte to support a later stage of infection, either uncoating or replication. This assertion was substantiated by the infectious center data shown in Table 2. Although virus was capable of gaining entry into the unstimulated normal MNL, this leukocyte inactivated the virus, and few infectious centers were formed. A productive infection, rather than inactivation of the virus, occurred within PHA-stimulated or HCL cells, and the number of infectious centers produced was much greater. Quantitative consideration of the data indicated that only a small fraction of the HCL cells participated in virus production. Similar results were found with PHA-stimulated normal MNL. When 10 cells were exposed to an excess of virus, only 1000 (0.1%) produced infectious virus (Table 2). However, FA examination of these cells demonstrated HSV antigens in 6 to 16%, and 6 to 8% of the cells showed ultrastructural evidence of virus replication (Table 1). These data suggest that the majority of the HCL cells do not become infected when exposed to HSV; of those that do become infected, only a small percentage produce infectious virus. The exact percentage of abortively infected cells is uncertain since cell-to-cell spread of virus may have increased the number of infected cells measured by electron microscopy or FA methods. A point that remains unclear is why hairy cells, in contrast to other leukemic cells, were capable of supporting the growth of HSV. One possibility would be that the ability to grow HSV is influenced by the series (monocytic, B-lymphocytic, or T-lymphocytic) from which the malignant cell arose. At present, a dispute exists concerning the clonal origin of the malignant cell in HCL. Some investigators have presented evidence
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for a monocytic origin,32 whereas others have data supporting a B-l-mphocytic origin of this leukocvte.3-35 In the murine system, HSV has been shown to be incapable of productively infecting mitogen-stimulated T lymphocytes." Murine monocvtes and blast-transformed B lymphocytes. how ever, are able to replicate infectious virus. 3 3 Theoretically, either monocvtes or B lymphocytes could have the potential for replicating HSV. Howvever, if HCL cells are of the B-lvmphocvtic series, as most data now suggest, then the type of cell alone could not account for the ability of the leukocvte to grow virus, since replication of HSV was not observed in CLL or LSCL cells which had B-cell characteristics. Other factors, perhaps unique to HCL cells, such as metabolic activity or degree of differentiation, must also influence the ability of leukemic cells to replicate HSV. References 1. Berg RB: Multiplication of Echo 9 virus in suspensions of human leukocytes. Proc Soc Exp Biol Med 108:742-744. 1961 2. Gresser I. Chany C: Multiplication of poliovirus type I in preparations of human leukocytes and its inhibition by interferon. J Immunol 92:889-893. 1964 .3. Edelman R, Wheelock EF: V'esicular stomatitis virus replication in human leukocvte cultures: Enhancement by ph%tohemagglutinin. Science 154:1053-1055. 1966 4. Edelman R, Wheelock EF: Specific role of each human leukocyte type in viral infections. I. Monocvte as host cell for vesicular stomatitis virus replication in vitro. J V'irol 1:1139-1149, 1967 3. Miller G, Enders JF: Vaccinia virus replication and cytopathic effect in cultures of phv-tohemagglutinin-treated human peripheral blood leukocvtes. J Virol 2:787-792. 1968 6. Wheelock EF. Edelman R: Specific role of each human leukocvte type in viral infections. III. 17D yellow fever virus replication and interferon production in homogeneous leukocv-te cultures treated w-ith phvtohemagglutinin. J Immunol I a3:429-436, 1969 7. Lambriex NM. van der Veen J: Comparison of replication of adenovirus type 2 and type 4 in human lymphoc\-te cultures. Infect Immun 14:618-622. 1976 8. Edelman R. Wheelock EF: Specific role of each human leukocyte type in viral infections. II. Phytohemagglutinin-treated lymphocytes as host cells for vesicular stomatitis virus replication in citro. J Virol 2:440-448 1968 9. W%7illems FTC, Melnick JL, Rawls WVE: Replication of poliovirus in phvtohemagglutinin-stimulated human lymphocy%tes. J Virol :3:431-457, 1969 10. N.ahmias AJ, Kibrick S, Rosan RC: Viral leukocvte interrelationships. I. Multiplication of a DNA virus-herpes simplex-in human leukocv-te cultures. J Immunol 93:69-74, 1964 11. Bouroncle BA, Clausen KP. Damer EM: Replication of herpes simplex virus in cultures of phy-tohemagglutinin-stimulated human lymphocytes. J Natl Cancer Inst 44:1065-1078, 1970 12. Kleinman LF. Kibrick S, Ennis F. Polgar P: Herpes simplex virus replication in human lymphocyte cultures stimulated with phytomitogens and anti-lymphocyte globulin. Proc Soc Exp Biol Med 141:1095-1099. 1972 13. Henle VN7, Henle G. zurHausen H: Effect of herpes simplex v-irus on cultured Burkitt tumor cells and its failure to influence the Epstein-Barr virus carrier state. Cancer Res 29:489-494, 1969
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14. Floyd R, Glasser R. Vonka V, Benvesh-Melnick MI: Studies on the growth of herpes simplex virus in lvsmphoblastoid cells. Acta V'irol (Praha) 15:133-142, 1971 15. Gerber A, Sauter C. Lindenmann J: Fowl plaque virus adapted to human epithelial tumor cells and human mveloblasts in vitro. II. Replication in human leukemic mveloblast culture. Arch Ges Virusforsch 40:255-264, 1973 16. Muller SA, Herrrnann EC Jr. Winkelmann RK: Herpes simplex infections in hematologic malignancies. Am J Med 52:10'2-114. 1972 17. Cappel R, Klastersky J: Herpetic meningitis (type 1) in a case of acute leukemia. Arch Neurol 28:415-416, 1973 18. Habel K: Virus neutralization test. Fundamental Techniques in Virology. Edited by K Habel, NP Salzman. New York. Academic Press. Inc.. 1969, pp 291-293 19. Cohen HJ: Human lymphocyte surface immunoglobulin capping: Normal characteristics and anomalous behavior of chronic lymphocy-tic leukemic lymphocytes. J Clin Invest 55:84-93, 1975 20. Jarvis SC, Snyderman R. Cohen HJ: Human lymphocyte motility: Normal characteristics and anomalous behavior of chronic lymphocvtic leukemia cells. Blood 48:7 1 -729, 1976 21. Aisenberg AC. Wilkes B: Lymphosarcoma cell leukemia: The contribution of cell surface study to diagnosis. Blood 48:707-715. 1976 22. Catovskv D, Pettit JE, Galton DAG. Spiers ASD. Harrison CV: Leukemic reticuloendotheliosis ('Hairy' Cell Leukaemia). A distinct clinico-pathological entity. Br J Haematol 26:9-27. 1974 23. Hamper B, Notkins AL, Mage NIt, Keehn MA: Heterogeneity in the properties of 7-S and 19S rabbit-neutralizing antibodies to herpes simplex virus. J Immunol 100:386-593. 1968 24. Ship II. Ashe WK, Scherp HW: Recurrent "fever blister" and "canker sore." Tests for herpes simplex and other viruses with mammalian cell cultures. Arch Oral Biol 3:117-124. 1961 23. Shinkai K: Plaque morphology of herpes simplex virus in various cells under liquid overlay as a marker for its type differentiation. Jpn J Microbiol 19:459-462. 1973 26. Ashe WVK. Scherp HN: Antigenic analysis of herpes simplex virus by neutralization kinetics. J Immunol 91:658-6653 1963 27. Notkins AL. Rosenthal J. Johnson B: Rate-zonal centrifugation of herpes simplex virus-antibody complexes. Virology 4.3:321-325. 1971 28. Bvurum A: Isolation of mononuclear cells and granulocvtes from human blood. Scand J Clin Lab Invest 21 (Suppl 97):77-89. 1968 29. Theofilopoulos AN. Brandt WVE. Russell PK. Dixon FT: Replication of dengue-2 virus in cultured human lymphoblastoid cells and subpopulations of human peripheral leukocvtes. J Immunol 117:953-961, 1976 .30. Porath J, Aspberg K. Drevin H, Axen R: Preparation of cyanogen bromide activated agarose gels. J Chromatogr 86:33-36. 1973 31. Sinkovics JG. Gy6rkey F. Gvorkev- P: Leukemic reticuloendotheliosis and "hairy cells" 293:1016. 1976 32. Scheinberg NI. Brenner Al. Sullivan AL. Cathcart ES. Katavama I: The heterogeneity of leukemic reticuloendotheliosis. "hairy cell leukemia." Evidence for its monocytic origin. Cancer 37:1302-1307d 1976 .33 Deegan NIJ, Cossman J. Chosney BT. Schnitzer B: Hairy cell leukemia. An immunologic and ultrastructural study. Cancer '38:1932-1961. 1976 34. Utsinger PD. Yount WJ, Fuller CR. Logue NIJ, Orringer EP: Hairy cell leukemia: B-lymphocyte and phagocytic properties. Blood 49:19-27. 1977 .35. Haak HL. deNMan JCH. Hijmans WV, Knapp XV. Speck B: Further evidence for the lvmphocytic nature of leukaemic reticuloendotheliosis (hairy cell leukaemia). Br J Haematol 27:31-.38, 1974
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36. Kirchner H, Hirt HM, Kleinicke C, Munk K: Replication of herpes simplex virus in mouse spleen cell cultures stimulated by lipopolysaccharide. J Immunol 117:1753-1756, 1976 37. Johnson RT: The pathogenesis of herpes virus encephalitis. II. A cellular basis for the development of resistance with age. J Exp Med 120:359-374, 1964 38. duBuy H: Effect of silica on virus infections in mice and mouse tissue culture. Infect Immun 11:996-1002, 1975
Acowledgmnts Excellent technical assistance was provided by Mrs. Sylvia LeGoff and Mr. Thomas Lassater. We also wish to thank Mrs. Frances Slocum for manuscript preparation.
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IA-Electron IIAmicrograph of an HSV-infected MNL from a patient with hairy cell leukemiaa after 3 days of)f culture. cufture. Fqu Fe" At the periphery of the cell, microvillous structures can be seen. The contains numerous imerous fat
cytoplasm osmiophilic 3philic per i droplets, matin is clumped mitochondria, and dilated endoplasmic reticulum. Within the nucleus, chromatin )ed at the droplet swollen nuclear mi membrane, and several clusters of nucleocapsid structures can be seen adjacent to this his aggregated Ichromatin (arrows). Je the cell an enveloped (arri (arrom B-Two herpesvirus capsid structures can be seen in the nucleoplasm and outside -nveloped virus particle is present. (Uranyl magnesium acetate and lead citrate, A x 16,800; B x 105,000) pparti '000)
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Arnerican Joumal of Pathology
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