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Journal o f General Virology (1992), 73, 3275-3279. Printed in Great Britain 3275 Murine gammaherpesvirus 68 establishes a latent infection in mouse B lymphocytes in vivo N. P. Sunil-Chandra,* S. Efstathiou~ and A. A. Nash Department o f Pathology, University o f Cambridge, Tennis Court Road, Cambridge C B2 1 Q P, U.K. Murine gammaherpesvirus 68 (MHV-68) is able to persist in spleen cells of infected mice. To determine the cell type harbouring persistent virus, spleen cells from infected animals were separated into immunoglobulin (Ig)-positive (B cell-enriched), Ig-negative (T cellenriched) and plastic-adherent (macrophage-enriched) fractions. These cells were co-cultivated with permis- sive BHK-21 cells in an infectious centre assay. The consistent recovery and enrichment of infectious centres in the Ig-positive fraction clearly demonstrates that B cells are a major site of virus persistence/latency. This observation indicates that MHV-68 is biologically similar to Epstein-Barr virus and other members of the B cell lymphotropic gammaherpesvirus 1 subgroup. Gammaherpesviruses are generally considered to be lymphotropic in nature, with the ability to establish latent infections within lymphocytes (Roizman et al., 1981; Honess, 1984). These viruses can induce a lymphoproliferative disease in the infected host and can efficiently immortalize lymphocytes infected in vitro, e.g. Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS) (Shope et al., 1973; Crawford et al., 1982; Fleckenstein & Desrosiers, 1982; Rickinson et al., 1989). On the basis of the available data, gammaherpesviruses have been subdivided into B cell-tropic (gammaherpesvirus 1), characterized by EBV and related viruses of old world monkeys and apes, and T cell-tropic (gammaherpesvirus 2), such as HVS and herpesvirus ateles, both infecting new world monkeys (Honess, 1984). This classification may not hold true for all gammaherpesviruses; for example, herpesvirus sylvilagus can establish a latent infection in both B and T cells of cottontail rabbits (Kramp et al., 1985). We have been studying murine herpesvirus 68 (MHV68), a naturally occurring murid herpesvirus originally isolated from bank voles (Clethrionomys graleolus) in Czechoslovakia (Blaskovic et al., 1980). Limited sequence analysis of the MHV-68 genome has shown this virus to be closely related to the gammaherpesviruses of primates, EBV and HVS, in terms of both its gene content and organization (Efstathiou et al., 1990a, b). However, the overall genome structure and G + C content of MHV-68 are most similar to those of the gammaherpesvirus 2 group. Studies on primary infection of BALB/c mice have shown the lung to be the main tissue productively infected by MHV-68, with virus present in alveolar epithelium and mononuclear cells (Sunil-Chandra et al., 1992). As with the other gammaherpesviruses, the spleen appears to be the major site of virus persistence, with latently infected cells detected by a co-cultivation assay. This technique has been used widely to detect latently infected lymphoid cells taken from animals infected with HVS (Falk et al., 1972; Rabson et al., 1971) and herpesvirus sylvilagus (Kramp et al., 1985; Medveczy et al., 1984). The ability to recover virus by explant culture, but not by direct homogenization of spleen, and the lack of virus antigen expression at this site at late times postinfection (p.i.) (Sunil-Chandra et al., 1992), is taken by us as a definition of virus latency. The aim of the present study was to identify the lymphocyte population harbouring latent MHV-68. To achieve this, spleen cells were separated into plasticadherent cells, to enrich for macrophages, immunoglobulin (Ig)-positive cells (B cells) and Ig-negative ceils (T cells) using anti-Ig-coated plates. This method offers a rapid and specific means of separating lymphocyte subpopulations (Nash, 1976; Mage et al., 1977; Mason et al., 1987; Wysocki & Sato, 1978). The number of cells harbouring latent virus from each subpopulation was determined by an infectious centre assay (Sunil-Chandra et al., 1992). In three separate experiments, 3- to 4-weekold BALB/c mice (Bantin and Kingman) were infected intranasally with 4 x 105 p.f.u. MHV-68 (Sunil-Chandra et al., 1992) and the spleen was removed on different days p.i. Spleen cells were separated into plastic-adherent, Igpositive (B cells) and Ig-negative (T cells) fractions, as shown in Fig. 1. Briefly, red blood cells (RBCs) were t Present address: Division of Medical Virology, Institute of Medical and Veterinary Science, Frome Road, Adelaide, SA 5000, Australia. 0001-1189 © 1992 SGM 3276 Short communication Spleen cell suspension Water lysis of RBCs IUncoated tissue culture platesI Supernatant 37 °C 1 h Adherent cells ............................ Sheep anti-mouse IgG-coated [ [ , tissue culture plates I Supernatant ~ :;:i:i;i;i:i:i: ~ ~i~ifraction~i~;;;~ ~i!i~;~!~i ~i~i:::::ii::i::!::ii::i::iiiiil ~ ................... :::~:re:n~ ~el:~s!:iliiiiii:i!iiii!i!ii:i:i:i:i:!ii:!i:i:i:i:!:i~ Bound cells ::::itfraction i~!!~iiiii!i::iiii:: ~i!il):~:~i~i~i:~:~i~i~i~!ii~i:::i:!::::::ii~ i:i:i:i!iii:i: Fig. 1. Flow diagram to illustrate the process involved in separating spleen lymphocytes using plastic dishes. lysed by resuspending spleen cells in 1 ml disilled water for 15 s and then in 10 ml of medium (RPMI 1640/20~ foetal calf serum). The cell suspension was washed once, counted and used in the lymphocyte experiments. Sheep anti-mouse I g G ( H + L ) afffinity-purified antibody (Sera Lab) was used to coat 60 mm tissue culture grade plastic Petri dishes (Falcon) for the positive selection of B cells by panning (Mage et al., 1977; Mason et al., 1987). Briefly, 4 ml antibody solution (10 p.g/ml) was added to a Petri dish for 16 to 18 h at 4 °C. The antibody solution was then decanted and the dish was washed with jets of PBS to remove any unbound antibody. PBS (5 ml) containing 0.2~ BSA was added to each plate and incubated for 30 min at room temperature to block unbound sites on the dish. Excess albumin was washed away and the dish was used for cell separation. Plastic-adherent cells were separated by incubating approximately 107 spleen cells in 5 ml medium in a 60 mm tissue culture grade Petri dish for 1 h at 37 °C in a humidified 5 ~o COz incubator. Non-adherent cells were resuspended and decanted. The cells present in 5 ml of medium were added to a sheep anti-mouse IgG(H + L)coated Petri dish and incubated for 2 h at 4 °C. The nonadherent, Ig-negative cells were resuspended and decanted for use as the T cell-enriched fraction. Dishes containing plastic-adherent cells and the Ig-positive cells were washed three times to remove any unbound cells. Finally, 2 x 106 BHK-21 cells in 5 ml medium were added to the plastic-adherent and B cell-enriched dishes, and also to the T cell-enriched fraction, and co-cultivated at 37°C in a 5 ~ CO, incubator. After 5 days the monolayers were fixed in 10~ formal saline, stained with 1~ toluidine blue and the number of infectious centres was counted. The results in Table 1 indicate clearly that MHV-68 could be recovered from the B cell-enriched fraction (Igpositive cells) and from the plastic-adherent cells early in acute infection (i.e. days 5 and 7 p.i.). However, towards the end of the acute disease and during the recovery period (days 10 to 36), a high proportion of infectious centres were recovered, predominantly from the B cellenriched fraction. The reduction in the number of infectious centres observed at day 36 may indicate that there is a loss of latent sites. This is supported by a previous observation on the number of infectious centres recovered at 90 days p.i. (Sunil-Chandra et al., 1992). This suggests that, following the acute infection, the immune system may regulate the number of latently infected spleen cells. In contrast, no or very few infectious centres were detected in the T cell-enriched fraction. From these studies it would appear that the principal cell harbouring MHV-68 during the acute and latent infection is an Igpositive B lymphocyte. The mean number of infected cells in the B cell population (Table 1) was found to be approximately 1/105 spleen white cells, which is similar to the number reported in patients with infectious mononucleosis but more than that seen in B cells taken from peripheral blood during a chronic EBV infection (Rocchi et al., 1977). To establish the percentage of macrophages, B and T lymphocytes present in unseparated spleen cells, and following separation on uncoated dishes or anti-Igcoated plates, flow cytometric analysis was carried out using fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (MAbs) specific for B cells (Ly5+), T cells (CD4 ÷ and CD8 ÷) and macrophages. MAbs Ly5 (B220)-FITC and Macrph-FITC (Coulter Immunology) were used at working dilutions of 1:20 in PBS. Ly5 (B220) is expressed on pre-B cells, B cells and some antibody-secreting cells including some plasma cell tumours (Kincade, 1987). The antibody is from clone RA36B2, rat IgG2b. MAb Macrph reacts with polypeptides specifically on macrophages and their precursors, and is derived from clone M/70, rat IgG2a. Rat antimouse CD4 and CD8 MAbs labelled with FITC were kindly provided by Dr S. Cobbold of this department, and used as a mixture at working dilutions of 1 : 100 and 1:200, respectively, in PBS. Sheep anti-rabbit IgGFITC (Wellcome Diagnostics) was used as the negative control antibody for direct immunofluorescence. In Table 2, spleen cells were separated into different populations as described, and stained with the various FITC-labelled antibodies. The adherent cell fractions were recovered for this purpose, either following incubation in 5 mM-glucose solution for 15 min and Short communication T a b l e 1. 3277 Identification of spleen cell types harbouring virus during acute and latent MHV-68 infection Time p.i. (days) Expt. 1 5 10 Expt. 2 20 Expt. 3 24 36 Total number of spleen white cells/spleen ( × 10-7) B cell-enriched population T cell-enriched population* Plastic-adherent cells t 2 3 2-5 4 1.1 12 18 0 0 0 0 58 4 36 4 352 288 282 1 0 1 0 0 18 13 0 25 32 0 26 10 16 Mouse no. 1 1 4 2 3 1 2 3 2.6 2.8 2.7 2.3 1.4 Infectious centres/10~ spleen white cells 1 9 190 2 2 3 4 5 6 9 9 9 9 2.5 166 256 224 88 101 0 2 0 1 6 38 23 51 46 6 12 1 2 3 8 9.5 8 6.6 5.2 6-6 106 227 168 84 49 27 1 1 1 6 7 17 0 10 0 0 0 0 1 2 3 * Cells non-adherent to anti-IgG(H +L)-coated plates. t Non-specifically adherent cells. T a b l e 2. Flow cytometric analysis of spleen cell subpopulations obtained using uncoated and anti-Ig-coated tissue culture plates Positive cells (~)* MAb Ly5 (B220)-FITC Anti-CD4-FITC and anti-CD8-FITC Macrph-FITC Total spleen cells B cellenriched T cellenriched Plasticadherent 48 49 74 26 3-5 88 57 24 6 6 6 14 * Ten-thousand cells were gated and the percentage of positive cells was calculated from total cells gated in regions corresponding to lymphocytes and monocytes. pipetting (plastic-adherent cells), or incubating with 2 ~ mouse serum for 30 min and pipetting (Ig-positive cells). Table 2 shows that 7 4 ~ of Ly5 + B cells were present in the Ig-positive fraction compared to 3-5~ in the Ignegative T cell fraction. Failure to achieve a higher percentage of B cells in the Ig-positive fraction is almost certainly due to damage to cells following removal from the plastic dishes (Mage et al., 1977; Wysocki & Sato, 1978). The detection of up to 57~ Ly5 + cells in the plastic-adherent fraction helps to explain the presence of infectious centres in these dishes. However, the number of infectious centres recovered from these dishes was less than would be expected from 57~ B cells, when compared to the numbers recovered from 7 4 ~ B cells. This suggests that a particular subset of B lymphocytes may harbour the latent virus. The possibility that macrophages harbour the virus in a latent form seems unlikely because following their enrichment on plastic dishes no concomitant increase in the number of infective centres was observed. To support the observation made above, we used another technique for the separation of Ig-positive B lymphocytes involving immunomagnetic beads (Kvalhelm et al., 1988, 1989). This method involves separating spleen cells obtained from mice 20 days after an intranasal infection. The spleen cells were coated with rabbit anti-mouse I g G ( H + L ) and mixed with sheep anti-rabbit IgG-coated magnetic beads (M-280 dynabeads; Dynal) according to the manufacturer's instructions. The numbers of cells in the magnetic bead-flee fraction and the bead/cell rosette fraction were counted, 3278 Short communication T a b l e 3. Recovery o f M H V - 6 8 from spleen B cells positively selected 20 days p.i. b.v using immunomagnetic beads Mouse no. Spleen cell fraction B cell-enriched;~ B cell-depleted spleen cells B cell-enriched B cell-depleted spleen cells Ly5 + cells as determined by flow cytometry (%)* Infectious centres/107 cells~ 77 11 106 7 74 10 36 5 * Ig-positive cells present in both B cell-enriched and -depleted spleen cell fractions were stained with Ly5 (B220)-FITC MAb, which binds Ly5 surface antigen. Ten-thousand cells were gated from each of these cell preparations during flow cytometry. Percentage positive cells was analysed in the region of lymphocytes and the cell/bead rosettes (7000 B cell-enriched and 2000 B cell-depleted from each mouse). t Number of infectious centres was determined from 107 cells of each of these separated spleen cell fractions. :~ Surface Ig-positive mouse spleen cells (B cells) were first allowed to bind with rabbit anti-mouse IgG(H + L). This mouse B cell-rabbit antiIgG complex was then positively selected by using sheep anti-rabbit IgG-coated magnetic beads (M-280 dynabeads). cells were co-cultivated with BHK-21 cells to assay infectious centres, and an aliquot from each fraction was used for flow cytometry and fluorescence microscopy. The results (Table 3) show that co-cultivation of the Igpositive fraction gives a greater number of infectious centres than Ig-negative spleen cells. These results are consistent with the relative percentages of Ly5+ cells in both the Ig-positive cell-enriched and -depleted populations, as determined by fluorescence-activated cell sorting (Table 3), and therefore supports the previous observation that MHV-68 is indeed a B cell-tropic gammaherpesvirus. The data reported are consistent with murine B lymphocytes, probably a subset, being the principal site for virus persistence. In contrast to herpesvirus sylvilagus and members of the gammaherpesvirus 2 subgroup, mouse T lymphocytes were not a target for MHV68. This suggests that, biologically, MHV-68 is more related to the gammaherpesvirus 1 subgroup, despite having a genome structure more similar to that of the gammaherpesvirus 2 subgroup. Further studies are needed to identify the virus receptor and the nature of B cells infected. In man, EBV enters B cells via CR2 (CD21) (Fingeroth et al., 1984). 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SHOPE, T., DECHAIRO, D. & MILLER, G. (1973). Malignant lymphoma in cotton topped marmosets after inoculation with Epstein-Barr 3279 virus. Proceedings of the National Academy of Sciences, U.S.A. 70, 2487-2491. SUNIL-CHANDRA, N. P., EFSTA~dIOU, S., ARNO, J. & NASI-I, A. A. (1992). Virological and pathological features of mice infected with murine gammaherpesvirus 68. Journal of General Virology73, 23472356. SVOBODOVA, J., BLASKOVIC, D. & MISTRIKOVA, J. (1982). Growth characteristics of herpesviruses isolated from free living small rodents. Acta virologica 26, 256-263. WYSOCKI, L. J. & SATO, V. L. (1978). "Panning" for lymphocytes: a method for cell selection. Proceedings of the National Academy of Sciences, U.S.A. 75, 2844-2848. (Received 29 June 1992; Accepted 5 August 1992)