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Journal of General Virology (1994), 75, 2329--2336. Printedin Great Britain 2329 The establishment of cytomegalovirus latency in organs is not linked to local virus production during primary infection Monika Balthesen, Liane Dreher, Pero Lu~in and Matthias J. Reddehase*t Department of Virology, Institute for Microbiology, University of Ulm, Albert-Einstein-Alice 11, 89081 Ulm, Germany Recovery from primary cytomegalovirus (CMV) infection is associated with resolution of the productive infection without clearance of the virus genome from affected organs. The presence of latent CMV genome in multiple organs provides the molecular basis for recurrence of CMV within multiple organs, and explains the diversity in the organ manifestations ofrecrudescent CMV disease during states of immunodeficiency. As a part of a unifying concept of multifocal CMV latency and recurrence, previous work has demonstrated the importance of primary virus replication for the overall load of latent CMV in organs and the risk of recurrence. In the present report, the establishment of CMV latency was studied in a murine model in which the course of primary infection in the immunocompromised host after syngeneic bone marrow transplantation was modulated Introduction Lymphohaematopoietic reconstitution of a cytolytic T cell response after bone marrow transplantation (BMT) is pivotal for the control of a post-transplantation primary or recurrent infection of patients with the human herpesvirus type 5 (HHV-5), human cytomegalovirus (CMV) (Quinnan et al., 1982; Reusser et al., 1991). Experimental studies in the murine model of CMV disease (for reviews, see Koszinowski et al., 1990, 1993) have identified CD8 + T cells as the principal antiviral effector subset of T lymphocytes that limits virus multiplication and tissue lesions in organs (Reddehase et al., 1985, 1987a, b, 1988). Recent clinical trials were aimed at supplementing an insufficient CD8 + T cell reconstitution in patients after BMT and HHV-5 infection by adoptive transfer of CMV-specific CD8 + T cell lines (Riddell et al., 1992). The establishment of latency after the resolution of primary infection is a characteristic of herpesvirus infections (Roizman & Sears, 1987), such as CMV t Present address: Institute for Virology, Johannes-GutenbergUniversity,Hochhausam Augustusplatz,55101 Mainz,Germany. 0001-2295 © 1994SGM by a CD8 + T cell immunotherapy. The antiviral CD8 + effector cells limited virus replication in all organs and protected the recipients from lethal CMV disease, but after resolution of the productive infection virus DNA remained. Interestingly, the copy number of latent virus DNA in tissue did not quantitatively reflect the preceding virus production in the respective organ. Specifically, in contrast to the case in the lungs and the salivary glands, virus replication in the spleen was suppressed by CD8 + T cells to below the limit of detection; yet, virus DNA was also detected in the spleen during latency and accordingly, virus recurrence in the spleen could be induced. These findings demonstrate that the control of virus replication in a particular organ does not prevent the establishment of latency in that organ. infection (for reviews, see Jordan, 1983; Mocarski et al., 1990). Latent murine CMV DNA has been detected in a variety of organs that are also the sites of productive infection and viral pathogenesis, including the spleen, lungs, salivary glands, heart, liver, brain, kidney and the adrenal glands (Klotman et al., 1990; Balthesen et al., 1993; Collins et al., 1993; Reddehase et al., 1994). We have shown previously that the copy number of latent virus DNA detected in tissues by PCR correlates with the risk of virus recurrence in the respective organ (Balthesen et al., 1993), and that virus replication and dissemination during primary infection determines the load of latent CMV (Reddehase et al., 1994). It is obvious that CD8 + T cells primed during the physiological immune response do not prevent CMV latency. However, it is an essentially different question, so far unanswered for CMV, whether or not a timely provision of terminally differentiated antiviral CD8 + effector cells by cell transfer can clear the viral DNA. Whereas the resolution of productive infection by CD8 + T cells has been demonstrated for many different viruses (for a review, see Kaufmann & Reddehase, 1994), clearance of the viral genome has rarely been analysed. That CD8 + T cells are principally capable of clearing virus genome has been documented for the persistent Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 05 May 2017 00:38:43 2330 M. Balthesen and others Draining popliteal lymph node Replicates bp BALB/c - 363 100 No disease CMV s.c. /~4,_ J ,~,, 41 ~L~ :-Ot 8 days Immune response :~ ' CD8 - 363 ~'~ CTL-P c 1 ...... '~ 8 days > CTL e in culture plus IL-2 A " - 363 6 Gy Fig. 1. Sensitivity of the detection of a viral sequence by PCR. A 363 bp sequence of exon 4 of the ieI gene of murine CMV was amplified from 1, 10 and 100 molecules ofplasmid piE111 in 10 independent replicates each. The total amount of DNA per sample was adjusted to 3 lag with certified-negative carrier DNA from mouse spleen. Shown are the autoradiographs obtained after hybridization of the amplification products with a radioactively labelled internal oligonucleotide probe. ~ccCD4+C Adoptive transfer i.v. °1 .i °' L CMV s.c~- Bone marrow transplantation i.v. ~ CD8 infection of mice with lymphocytic choriomeningitis virus (Oldstone et al., 1986). By combining the approach of adoptive CD8 + T cell immunotherapy with the detection of viral DNA in organs after the resolution of primary infection we demonstrate here that a dose of CD8 + T cells that suffices for protection against lethal disease and that prevents overt virus replication in the spleen does not preclude latent infection of the spleen. Methods Bone marrow transplantation, infection and cytoimmunotherapy. Recipients as well as donors for syngeneic BMT were specifiedpathogen-free, 8-week-old female mice of the inbred strains BALB/c or BALB/c-H-2 dm~ (dm2). The dm2 strain is a mutant of BALB/e, in which a region of the major histocompatibility complex (MHC), encompassing the gene encoding the MHC class I glycoprotein Ld, is deleted (Klein et al., 1983). For haematoablative conditioning, BMT recipients were total-body ),-irradiated with a single dose (6 Gy) from a caesium-137 source (OB58; Buchler) delivering a dose rate of 0.708 Gy/min. Donor femoral bone marrow cells (BMC) were obtained as described previously (Mutter et al., 1988). BMC (1 × 106) were infused into the tail vein of recipients about 6 h after the irradiation. Infection with 1 × l0 b p.f.u, of murine CMV, strain Smith (ATCC VR194) was performed subcutaneously at the left hind footpad about 2 h after BMT. In the case of cytoimmunotherapy, 1 × 10~ antiviral T cells of the CD8 + subset were infused intravenously simultaneously with the BMC. The CD8 + T cells were prepared as described previously (Reddehase et al., 1987b). In brief, BALB/c mice were infected at the left hind footpad and, 8 days later, sensitized T lymphocytes obtained from the draining popliteal lymph node were propagated in microcultures for a further 8 days in the presence of recombinant interleukin (IL)-2, but with no viral antigen added. CD4 + T cells were eliminated from the polyclonal, short term CD4 + and CD8 + T cell line by treatment with monoclonal antibody (MAb) GK-1.5 and rabbit complement. Read out: survival control of infection clearance of CMV genome Fig. 2. Experimental regimen for CD8 ÷ T cell supplementation therapy. Antiviral T lymphocytes were sensitized by subcutaneous (s.c.) infection of BALB/c mice with murine CMV. Cells derived from the draining lymph node were propagated in culture in the presence of IL-2. During this period, cycling cytolytic T lymphocyte precursors (CTL-P °) develop into mature effector cells (CTLe). CD4 ÷ T cells were eliminated from the short term T cell line by treatment with anti-CD4 antibody and complement (c~CD4+ C). The purified CD8 + T cells were mixed with BMC and infused intravenously (i.v.) into ),-irradiated (6 Gy) recipients. The steps in the purification of primed CD8 + T cells were monitored by two-colour cytofluorometric analysis by using fluorochrome-conjugated antibodies specific for the marker molecules CD4 and CD8. The scales show fluorescence intensities. Contour lines represent cell frequencies in a 70% log mode. Numbers in the plot corners give the percentage of cells located in the indicated quadrants. Cytofluorometric analys&. Two-colour cytofluorometric analysis was performed with a FACScan (Becton Dickinson) using LYSIS (Becton Dickinson) software for computer analysis. Two-dimensional contour plots represent 25000 cells. Contour lines show cell frequencies in a 70% log mode. Single-fluorescence histograms represent 10000 cells. O) CD-phenotyping o f T cells. Lymph node ceils as well as cells of the T cell line were stained with the directly fluorochrome-conjugated MAbs rat anti-mouse CD8-fluorescein isothiocyanate (FITC) (clone 53-6.7; Becton Dickinson) and rat anti-mouse CD4-phycoerythrin (PE) (clone SK3; Becton Dickinson). (ii) Tracking of donor CD8 + T cells in M H C chimeras. Cells from the spleen (enriched for T cells using nylon wool), were stained with three antibodies: mouse MAb anti-L d (clone CL 9011-A; Cedarlane), FITC-conjugated goat anti-mouse IgG2a (Medac) and MAb rat antimouse CD8 PE (clone 53-6.7; Boehringer Mannheim). CD8 + T cells were selected analytically by the setting of an electronic window on cells with positive PE fluorescence. Determination o f virus titres and induction of i n vivo recurrence. Virus titres in organ homogenates during acute infection were determined by Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 05 May 2017 00:38:43 Local C M V production and latency Survival rate 2331 Virus replication in organs (a) I I [ I I l I 100% -7 £ 50% - 6 - 5 4 37 0% 2 m L (b) I I I 2 3 (weeks) 4 I I "-"7"-~-- I T I l I l I F"- 4 5 ~c ~ 0 100% 50% 0% 0 1 0 1 2 3 (months) Time after infection Fig. 3. Time course of CMV infection and disease. Syngeneic BMT was performed with BALB/c as donor and recipient strain. (a) No therapy; (b) therapy with 1 × l0 s BALB/c CD8 ÷ T cells. Left panel: survival rates (n = 20). Right panel: virus replication in organs determined by plaque assay from organ homogenates. Symbols represent virus titres in three individuals per time point. Vertical bars indicate the range, horizontal bars mark the median values. DL, Detection limit of the standard plaque assay. Cases in which the respective organ was negative for all three tested individuals are depicted only on the first occasion in the kinetics. The asterisk indicates that the organ homogenate was retested with the highest sensitivity plaque assay capable of detecting 1 p.f.u, per organ. Symbols: (O), salivary glands; (©), lungs; (ll), spleen. a standard 4 day plaque assay on permissive mouse embryo fibroblasts essentially as described previously (Reddehase et al., 1985). The sensitivity of detection was increased 20-fold by using centrifugal infection. Testing of an aliquot of 1% of the homogenate thus defines the detection limit of this assay as 100 p.f.u, per organ. To ensure the absence of infectious virus during latent infection, the sensitivity was increased to 1 p.f.u, per organ by plating all of the homogenate. Further, the plaque assay was then performed without semi-solid overlay to allow any secondary plaque formation. A negative score is assigned to organs if no plaque was detected after 4 days as well as 10 days of the assay. Recurrent infection in vivo was induced by )~irradiation (6 Gy) of latently infected mice. Recurrent virus was detected 14 days later with the highest sensitivity plaque assay. Inactivation of murine CMV was done by irradiation with u.v. light of wavelength 254 nm. Its efficacy was monitored by the highest sensitivity plaque assay, and was verified by the absence of infection after inoculation of y-irradiated mice. hybridization with a 32P-end-labelled internal oligonucleotide probe. Autoradiographic signal intensities were found to increase linearly only between 10 and 100 copies of the test sequence (Balthesen et al., 1993). The sensitivity of detection was determined by the amplification of 100, 10 and 1 molecules of piE111 in 10 independent replicates (Fig. 1). The signal variance seen with 10 template molecules reflects the statistical variance given by the Poisson distribution. From the fractions of negative replicates, the most probable number of detectable templates is calculated as being four with a 95 % confidence interval of 2 to 8 (maximum likelihood method). Detection o f vital and cellular D N A sequences. Organs and blood leukocytes were processed for DNA isolation, and a 363 bp sequence from exon 4 of the murine CMV immediate early (IE) gene iel as well as a sequence from the mouse fl-actin gene were amplifed by PCR in 35 cycles as described previously (Balthesen et aL, 1993). Plasmid piE111, which encompasses the iel gene of murine CMV (Smith strain), served as the positive control. Amplification products were analysed by agarose gel electrophoresis, Southern transfer and Supplementation of B M C with mature, antiviral CD8 + T effector cells was performed in syngeneic experimental B M T to modulate the course of a concurrent primary murine C M V infection. Recipients of B M T and donors of B M C as well as of CD8 + T cells were B A L B / c mice ( M H C haplotype H-2d). The number of B M C was adjusted so as to provide full protection against the Results Protective and antiviral efficacy of CD8 + T cell supplementation therapy Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 05 May 2017 00:38:43 2332 M. BaIthesen and others radiation-mediated haematological aplasia, but to be insufficient for controlling C M V infection (Mutter et al., 1988). This strategy was chosen as a model for clinical trials of a CD8 + T cell supplementation therapy in patients with a failure in endogenous T cell reconstitution after B M T (Riddell et at., 1992). The experimental regimen for B M T and cytoimmunotherapy of C M V infection is outlined in Fig. 2. The steps in the generation and purification of polyclonal, antiviral CD8 + T effector cells were monitored by two-colour cytofluorometric analysis of the expression of CD4 and CD8 cell surface molecules. The CD8 + T cell supplementation therapy was clearly beneficial in that it significantly reduced mortality between the first and third week after B M T and infection (Fig. 3) as well as wasting syndrome morbidity (daily inspection, not shown). This protection was accompanied by a 100-fold (lungs) to 1000-fold (salivary glands) reduction of virus titres at the peak time of mortality, that is at around 2 weeks (Fig. 3). The control of infection in the spleen is particularly efficient. Although the spleen is a prominent target organ for C M V after B M T (Fig. 3 a), virus replication at this site was suppressed by antiviral CD8 + T cells to below the limit of detection (Fig. 3b). As this limit was 100 p.f.u, in the standard virus plaque assay, the spleens were then retested by plating all of the homogenate in the highest sensitivity plaque assay. No p.f.u, were detected in spleen homogenates at 1, 2 and 4 weeks after CD8 + T cell transfer. As the mice are negative for antibodies to C M V early after BMT, the assay was not influenced by virus neutralization. After 4 to 5 months, acute infection had resolved in all tested organs and in both experimental groups. Adoptively transferred CD8 + T cells are recruited to a target site o f viral pathogenesis and persist long term in the recipients Since the effect of CD8 + T cell transfer was most pronounced in the first weeks after infection and was most prominent in the spleen, we tested whether the transferred cells were indeed recruited to that site to perform their function. A chimeric model was used to distinguish transferred donor CD8 + T cells from recipient CD8 + T cells regenerated during lymphohaematopoietic reconstitution. D o n o r cells for the immunotherapy were again the BALB/c-derived antiviral CD8 + T cells used in the preceding experiment, whereas syngeneic B M T was performed in the L d gene deletion mutant B A L B / c - H 2 am~, in which the M H C class I glycoprotein L ~ is not expressed. The fate of the donor cells within the recipients was tracked by cytofluorometric detection of L d on the surface of CD8 + T cells (Fig. 4). It should be noted that Time p.i. (weeks) 4 2 90% 8 35% 21% Expression of L~t(log10fluorescence intensity) Fig. 4. Fate of transferred donor-type CD8÷ T cells in the recipient. Syngeneic BMT was performed with the La gene deletion mutant BALB/c-H-2din2 as donor and recipient strain. La-positive CD8+ T cells for the immunotherapy were derived from BALB/c mice. The origin of CD8÷ T cells in the chimeras was determined by two-colour cytofluorometric analysis by using antibodies specificfor CD8 and La. An electronic window was set to select for CD8+ T cells. The distribution of recipient (Ld-negative)and donor (La-positive)CD8+ T cells is shown by single-fluorescencehistograms. Horizontal bars mark the donor cell fluorescence. Numbers in the upper right corners givethe percentage of donor-derived cells among the CD8÷ T cells. antiviral CD8 + T cells of these two mouse strains are cross-protective (not shown), which shows that protective immunity is not restricted to antigenic peptides presented by L d (for review, see Koszinowski et al., 1992). At 2 weeks after the transfer, most of the CD8 + T cells recovered from the spleen were of donor origin, whereas endogenous reconstitution of recipient CD8 + T cells became effective between 2 and 4 weeks posttransfer. Yet, donor cells were replaced only slowly and remained detectable even after 2 months (Fig. 4). That transferred CD8 + T cells migrate to target sites and can persist long term in the recipients is in accordance with previous reports (Cheever et al., 1986; Reddehase et al., 1988; Jamieson & Ahmed, 1989; Riddell et al., 1992). As the peak of mortality is at around 2 weeks, the main benefit of therapy by donor CD8 + T cells is to bridge the interim between haematoablative treatment and haematopoietic reconstitution, whereas termination of persistent infection is likely to be mediated mainly by the recipient's own cells. Load of viral genome in blood leukocytes during acute infection Acute infection is expected to be accompanied by blood cell-associated viraemia and P C R - D N A positivity of blood leukocytes. Viraemia was tested by a limiting dilution infectious centre assay that was performed by plating graded numbers of blood leukocytes in 24 replicates in flat-bottom well microcultures with m o n o layers of mouse embryo fibroblasts. The frequency of leukocytes positive for infectious virus was determined from the fractions of plaque-negative cultures on the Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 05 May 2017 00:38:43 Local C M V production and latency Controls 1¢ 1 21 (a) Blood leukocytes ] 0 1 2 3 4 t oO No therapy - 363 bp t CD8 t- therapy (b) 1¢ I 211 2 3 4 5 6 363 bp I - 363 bp (c) Blood cell D N A I 1 2 3 4 5 I - fl-actin Fig. 5. Detection of viral D N A in blood leukocytes during acute infection. (a) A 363 bp sequence from exon 4 of gene iel of routine CMV was amplified by PCR from blood leukocyte D N A isolated at 1 month p.i. and BMT with or without CD8 + T cell supplementation therapy. Leukocytes were titrated (10-fold dilutions) from 104 cells to 1 cell (lanes 4 to 0, respectively) before the D N A was isolated. Throughout, the PCR was run with 3 pg of DNA. For cell numbers ~< 105, the amount of D N A was adjusted with certified-negative carrier D N A from the spleen of an uninfected mouse, for cell numbers > 105 a respective aliquot of the D N A was tested. Arrows mark the autoradiographic signal that gives the frequency of positive leukocytes. Lanes ~b, contain all reagents except DNA. Control lanes 1 and 2 contain 10 and 100 copies, respectively, of plasmid piE111 adjusted to 3 gg with carrier DNA. (b) Blood leukocytes were titrated for D N A isolation and PCR at 1 month after inoculation of mice with a dose of inactivated virus that corresponds to l0 b p.f.u, o f infectious virus. Lanes 2 to 6 represent 100 to 106 cells in increasing 10-fold steps. (e) A sequence of the cellular fl-actin gene was detected by PCR in fractions of the D N A isolated from l0 s leukocytes from the experiment shown in (b). Lanes 1 to 5 contain 60 pg to 600 ng of D N A in increasing 10-fold steps with no carrier DNA. basis of the Poisson distribution by using the maximum likelihood method of calculation. In the group with no CD8 + T cell supplementation therapy, the frequency was 213 per 106 (i.e. about 1 per 5000) leukocytes at 4 weeks (95 % confidence interval: 149 to 306 per 106 leukocytes; P = 0.62). In contrast, after therapy, the number of positive leukocytes was below the detection limit. The presence of viral DNA in blood leukocytes was tested by PCR amplification of a 363 bp sequence from 2333 exon 4 of the iel gene of murine CMV (Balthesen et al., 1993). We have shown in a previous report that murine CMV DNA is associated with all subpopulations of blood leukocytes, with the highest relative frequency within Gr-I+CDllb ++ granulocytes (Balthesen et al., 1994). In Fig. 5(a), the frequency of blood leukocytes carrying viral DNA was determined at 4 weeks after BMT and infection. With 1 per 10 leukocytes, this frequency was much higher than the frequency of cells containing infectious virus and, surprisingly, there was no notable difference between the groups. An explanation could be that leukocytes that passively carry viral DNA are exempt from immune control and are saturated in both groups of mice. It should be considered that the high virus production in salivary glands (Fig. 3) probably does not contribute to viral DNA load in the blood, because virus produced in acinar glandular epithelial cells of the salivary glands is secreted via the salivary duct (Hensen & Strano, 1972; Jonji6 et al., 1989). That DNA-PCR positivity of blood leukocytes requires replicative CMV is documented in Fig. 5(b). Inoculum DNA from the same nominal dose of u.v.inactivated CMV was not detected in up to 106 blood leukocytes at 4 weeks after transfer. Amplification of a cellular sequence, namely a sequence of the mouse flactin gene (Balthesen et al., 1993), from the same DNA preparation assured that the PCR was working correctly (Fig. 5c). Cytomegalovirus latency and recurrence in organs The cellular site and the molecular nature of cytomegalovirus latency are still unknown. We therefore use the term 'latency' in its operational definition, namely the absence of detectable infectious virus in the presence of functional viral genomes capable of reactivation (Roizman & Sears, 1987). This definition is generally applied also for CMV latency (for a review see Jordan, 1983). At 1 year, that is about 8 months after the resolution of acute infection (Fig. 3), the absence of infectious virus was verified in three-quarter fractions of salivary gland, lung and spleen tissue of individual survivors of the CD8 + T cell therapy group by the highest sensitivity virus plaque assay. However, since putative virus replication at any other untested site necessarily escaped detection, we introduced absence of viral DNA from blood leukocytes as an additional criterion for a more stringent definition of CMV latency in organs. As shown in Fig. 5, acute infection is reflected by the presence of viral DNA in a high proportion of blood leukocytes, and this fact is used in clinical tests as a diagnostic marker for acute primary or recurrent CMV infection in patients (Chou, 1993). In a previous manuscript it was shown that murine CMV DNA is Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 05 May 2017 00:38:43 2334 M . Balthesen and others (a) Controls I~ 1 ° 211 I a II b II a III b ID -- 363 bp (b) [ ~b 1 21[ a b c d e f [ e~ II III O~ II III group analysed at 1 year (Fig. 6a; I to III), CMV DNA was virtually cleared from the blood. The two samples tested for each individual represented the DNA from 106 blood leukocytes. The yield of blood leukocytes and sensitivity of the PCR give an estimate of < 20 copies of viral DNA in total. Thus, there was no evidence for a low-level persistent infection at any site, and also a significant contamination of organ material by CMV DNA-positive blood leukocytes was practically excluded. In contrast, organs of the same three mice harboured viral DNA with copy numbers of/> 100 (salivary glands and lungs) or 10 to 100 (spleen) per 3 gg sample of organ DNA, with almost no variance between six samples tested, and with only little variance between the individuals (Fig. 6b). It should be noted that essentially the same result was obtained for individual mice with no CD8 ÷ T cell therapy (not shown). However, a quantitative comparison is unwarranted, because the few survivors after no therapy (Fig. 3 a) represent a highly selected group with the most efficient endogenous reconstitution of CD8+ T cell control. As a 3 lag sample of organ DNA represents the DNA of about 5 x 105 diploid cells, 100 copies per tested sample represent a load of approximately 200 viral genomes per 106 tissue cells. This estimate is close to the organ load observed in another murine CMV latency model, namely in latency established after the infection of newborn mice (Balthesen et al., 1993; Reddehase et al., 1994). In that model, the viral DNA load in organs was predictive of the risk of recurrence. In fair accordance with this previous finding, organ incidences of recurrence induced by y-irradiation (6 Gy) in 20 mice at 1 year after BMT, infection and CD8 ÷ T cell therapy were found to be 12/20 for the lungs, 11/20 for the salivary glands and 6/20 for the spleen. Discussion ~ ~ ~l~ia~ ~ ~l~i II III Fig. 6. Detection of viral DNA in organs during latency. Presence of the 363 bp viral test sequence in blood leukocyte and organ DNA was tested by PCR at 1 year after CD8 ÷ T cell supplementation therapy. Three mice were tested individually (I, II and III). Lanes a to f contain replicate 3 pg samples of DNA amplified independently. Control lanes are as in Fig. 5. maintained in blood leukocytes for an extended period after resolution of the acute infection, but is eventually cleared to below the limit of detection by PCR (Balthesen et al., 1993). In three individual mice of the therapy These data demonstrate the capacity of adoptively transferred donor CD8 + T cells to protect against lethal CMV disease after BMT in the period of immunological incompetence before haematological reconstitution becomes effective. However, although the peak virus replication was markedly reduced, CD8 + T cells had difficulties in terminating a low-level persistent infection in salivary glands and lungs, and they failed to clear the viral genome from solid tissues. This failure cannot be explained by a principal limitation of CD8 + T cell function, as experiments on another virus infection have shown that adoptively transferred CD8 + T cells can clear a viral genome from all organs (Oldstone et al., 1986). Hence, the observed maintenance of the CMV genome most likely reflects a property of this virus to escape immune control. Recent work on the regulation of Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 05 May 2017 00:38:43 Local C M V production and latency murine (Del Val et al., 1992) and human (Gilbert et al., 1993) CMV antigenic peptide presentation in permissive cells in vitro has identified molecular mechanisms of evasion from recognition by CD8 + T cells. However, as the described evasion is operative through viral genes expressed during the productive cycle and results in completion of the productive cycle, it can explain the persistence of virus production but not latency. Our data indicate that persistence and latency are indeed unlinked phenomena, as latency was found to be established in organs in which persistence occurred, namely in salivary glands and lungs, but also in the spleen where productive infection was prevented by the transfer of CD8 + T cells. A discrepancy between local virus production during acute infection and the amount of latent viral DNA in particular organs was also observed in our previous study, in which we compared latency established after primary infection at adult age with that established after neonatal infection (Reddehase et at., 1994). Specifically, after infection of neonates, the high and long-lasting virus production in the salivary glands was not reflected by an accordingly high load of latent CMV in salivary gland tissue. After infection of adults, virus production was restricted to the salivary glands and was undetectable in the lungs. Nonetheless, during latency, the lungs harboured more viral genomes than did the salivary glands. A similar dissociation between local virus production during acute infection and latency phenotype was mentioned in a review by Mocarski et al. (1990) for mutants of CMV. Thus, mutant RM 408 is debilitated for establishing latency in the spleen, even though it replicates well there. Conversely, this mutant becomes latent in the salivary glands, even though it is debilitated for salivary gland growth during the acute phase of the infection. However, that a certain overall level of virus replication and dissemination during primary infection is required to reach all target cells for latent infection was indicated by the marked difference in the organ load of latent CMV and the risk of recurrence between mice infected as neonates or as adults (Reddehase et al., 1994). The cellular site(s) of CMV latency in the various organs has not yet been identified. Analysis is made difficult by the low copy number of latent genomes in tissues, which predicts for the lungs a maximum frequency of one latently infected cell per 5000 tissue cells (Balthesen et al., 1993). For the spleen, the sinusoidal lining cell of the stroma was proposed as a candidate, because this cell type supported virus replication in acute infection and because latent virus was detected in a stromal fraction of the spleen (Mercer et al., 1988; Pomeroy et at., 1991). In contrast, the observed dissociation between local virus production and latency could indicate that the latently infected cells are not 2335 stationary tissue cells, but are immigrants derived from an organ site at which virus replication had occurred in the acute phase of the infection. An alternative, and probably more straightforward, explanation is that cells which account for the gross virus production during acute CMV infection are distinct from the cells in which latency is established, as is the case for herpes simplex virus (Roizman & Sears, 1987). The fact that productive CMV infection is cytocidal and causes histological lesions in affected organs further argues against an establishment of latency in productively infected cells. We propose that in its basic principles fl-herpesvirus latency is related to ~-herpesvirus latency. This work was supported by grants to M.J.R. by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 322, and by the Bundesministerium fiir Forschung und Technologie, project 01KE8817/0. References BALTHESEN,M., MESSERLE,M. & REDDEHASE,M. J. (1993). Lungs are a major organ site of cytomegalovirus latency and recurrence. Journal of Virology 67, 5360-5366. BALTHESEN, M., SUSA, M., LUC1N, P. & REDDEHASE,M.J. (1994). Cytomegalovirus DNA detected in blood leukocytes after resolution of productive infection does not originate from latently infected hematopoietic stem cells in the bone marrow. Croatian Medical Journal 35, 19-25. CHEEVER, M.A., BRITZMAN THOMPSON, D., KLARNET, J.P. & GREENBERG,P. D. 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