Dissemination and Replication of MCMV After Supraciliary Inoculation in Immunosuppressed BALB/c Mice

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1 Disseminatin and Replicatin f MCMV After Supraciliary Inculatin in Immunsuppressed BALB/c Mice Yanping Duan,* Zhnghua Ji,^ and Sally S. Athertn* Purpse. T study replicatin and disseminatin f murine cytmegalvirus (MCMV) in immunsuppressed (IS) and nn-is BALB/c mice after cular inculatin via the supraciliary rute. Methds. BALB/c mice were immunsuppressed by injectins f methylprednislne, and MCMV was injected via the supraciliary rute. Ocular and nncular tissues frm bth IS and nn-is mice were studied by plaque assay f tissue hmgenates. The frequency f virus-psitive leukcytes was determined by PCR. Results. In the inculated eye, virus replicatin was significantly higher in bth the anterir segment and the psterir segment f IS mice. Virus spread t extracular sites in bth IS and nn-is mice; hwever, significantly higher titers f virus were recvered frm the salivary glands and lungs f IS mice than frm nn-is mice, and clearance f virus frm these sites was delayed in IS mice. Virus spread frm the injected eye via leukcytes, and PCR amplificatin revealed that the frequency f virus-infected leukcytes was apprximately 200-fld higher in IS mice. Cnclusins. The results f these studies suggest that immunsuppressin significantly enhances virus replicatin in the inculated eye, salivary glands, and lungs, leads t a higher frequency f virus-psitive leukcytes, and delays clearance f virus frm cular and nncular tissues. These results als suggest that retinitis in the injected eye f IS mice crrelates with significantly higher titers f virus in the psterir segment. Invest Ophthalml Vis Sci. 1994;35: V-tytmegalvirus (CMV) retinitis is the mst frequently bserved phthalmic pprtunistic infectin in patients with acquired immune deficiency syndrme (AIDS). 1 " 3 Withut treatment, the disease results in blindness. Perhaps because f the relatively strict species-specificity f the virus, few animal mdels f CMV retinitis have been develped, and mst aspects f the pathgenesis f CMV retinitis remain t be deciphered. Frm the *Department f Cellular and Structural Bilgy, University f Texas Health Science Center at Sail Antni, Texas, and the ^Department f Micrbilgy and Immunlgy, University f Miami Schl f Medicine, Flrida. Presented in part at the Annual Meeting f the Assciatin fr Research in Visin and Ophthalmlgy, Sarasta, Flrida, May Supprted by Natinal Institutes f Health grants EY09169 (SSA) and EY02180 (cre grant, Department f Ophthalmlgy, University f Miami Schl f Medicine). Submitted fr publicatin fuly 2, 1993; revised September 24, 1993; accepted September 29, Prprietary interest categry: N. Reprint requests: Sally S. Athertn, Department f Cellular and Structural Bilgy, University f Texas Health Science Center, 7703 Flyd Curl Drive, San Antni, TX Recently, a murine mdel f CMV retinitis was described that shares sme features with human cytmegalvirus retinitis. 4 In this mdel, a high dse f the Smith strain f MCMV (5 X 10 4 plaque-frming units) injected via the supraciliary rute results in prgressive fcal necrtizing retinitis in nn-is BALB/c mice. In cntrast, inculatin f lw dse (5 X 10 2 PFU) f MCMV via the same rute int immuncmpetent BALB/c mice results in minimal retinal invlvement; hwever, injectin f the same dse f virus int IS mice results in acute retinitis and prgressin t retinal necrsis. 5 Cmparisn f the results in immuncmpetent and IS mice suggested that an intact hst immune system is required t prevent develpment f CMV retinitis after supraciliary inculatin f 5 X 10 2 PFU f MCMV in immuncmpetent mice. We hypthesized that an intact immune system prevents virus spread and replicatin within the inculated eye, spread f virus frm the inculated eye t 1124 Investigative Ophthalmlgy & Visual Science, March 1994, Vl. 35, N. 3 Cpyright Assciatin fr Research in Visin and Ophthalmlgy

2 MCMV Infectin in Immunsuppressed Mice 1125 nncular tissues, and virus replicatin in nncular tissues. In the experiments reprted herein, virus recvery and PCR were used t determine the amunt f virus in the inculated eye and extracular tissues and t detect MCMV DNA in bld cells, respectively. The results f these studies suggest that immunsuppressin facilitates hematgenus disseminatin f virus frm the inculated eye, increases viral replicatin in bth the inculated eye and nncular tissues, and delays clearance f virus frm cular and extracular sites. MATERIALS A METHODS Virus and Virus Titratin The virus used in these experiments was the Smith strain f MCMV, which was btained riginally frm Dr. Rbert Levy (Department f Micrbilgy and Immunlgy, University f Miami Schl f Medicine, Miami, FL). Virus stcks were prpagated frm salivary glands f MCMV-infected BALB/c mice as described previusly 4 and were titered by plaque assay n mnlayers f muse embry fibrblast (MEF) cells (Whittaker M.A. Biprducts, Walkersville, MD) grwn in tissue culture medium cntaining 10% fetal calf serum (HyClne, Lgan, UT). Virus stcks were stred at 70 C. The average titer f the virus ranged between 10 6 and 10 7 PFU/ml. A fresh aliqut f stck virus was thawed and diluted t the apprpriate cncentratin fr cular injectin immediately befre each experiment. Mice Female BALB/c mice 4 t 6 weeks f age (Tacnic Inc., Germantwn, NY) were used in all experiments. Mice were immunsuppressed by intramuscular injectin f 2.0 mg sterile methylprednislne acetate suspensin (Upjhn, Kalamaz, MI) every 4 days beginning n day 2. Cntrl mice were nt immunsuppressed. All mice were hused in accrdance with Natinal Institutes f Health guidelines, and all animal treatments in this study cnfrmed t the ARVO Reslutin n the Use f Animals in Research. Mice were maintained n a 12-hur light cycle alternating with a 12-hur dark cycle and were given unrestricted access t fd and water. All cular injectins were dne under sdium pentbarbital anesthesia (0.65 mg/10 g bdy weight). Inculatin Via the Supraciliary Rute Mice were anesthetized and inculated with 5 X 10 2 PFU f MCMV in the left eye via the supraciliary rute as previusly described. 4 Briefly, the left eye was prptsed, and the aqueus humr was remved by paracentesis. A superficial transscleral entry wund was made parallel and psterir t the limbus by intrducing the bevel f a sharp 30-gauge needle int the ptential supraciliary space. Tw micrliters f virus slutin were injected int the supraciliary space thrugh this wund with a blunted 30-gauge needle cnnected t a 250 n\ micrsyringe and an autmated pump (Hamiltn, Ren, NV). DNA Amplificatin Bld was cllected by cardiac puncture and anticagulated with sdium citrate slutin (Sigma, St. Luis, MO). Muse peripheral bld leukcytes (PBL) were separated frm whle bld using Lymphlyte-M fllwing the manufacturer's directins (Cedarlane Labratries Ltd., Hrnby, Ontari, Canada). Red bld cells were lysed in istnic ammnium chlride slutin. After washing with PBS, the leukcytes were cunted and frzen immediately at 70 C. Apprximately 1.25 X 10 4 and 2.5 X 10 6 (nn-is mice nly) cells f each sample were separately placed in 100 ^1 f ne-step lysis buffer (50 mm KC1, 10 mm Tris-HCl, ph 8.3, 2.5 mm MgCl 2, 0.1 mg/ml gelatin, 0.45% NP- 40, 0.45% Tween-20), briefly vrtexed, biled fr 10 minutes, and immediately cled n ice. The DNA lysates were cleared by brief centrifugatin at 13,000g, and the supernatants were stred at 20 C befre amplificatin. The PCR prtcl was a mdificatin f the methd f Saiki et al. 6 Tw MCMV primers and a prbe specific fr a 400-base-pair regin f an immediate-early gene 7 were synthesized by Clnetech (Pal Alt, CA). The MCMV sequences were 5'-CAGATC- TGAGTTTGATAATG-3' and 5'-TCATAGGCAATC- GTTTCCTG-3' fr 5' and 3' primers, respectively, and 5'-CATAGTGGCAGCCACCAACA-3' fr the prbe. The primers and prbe specific fr a sequence f the lng terminal repeat (LTR) f MMTV (murine mammary tumr virus) were derived frm a published sequence 8 and amplified a fragment f 165 base pairs. PCR amplificatin f the MLTR sequence was used t assess the amplifiability f the DNA extracted frm leukcytes befre amplificatin f MCMV sequences. The specificity f all primers and bth prbes has been described previusly. 910 PCR samples included 20 fxl f DNA lysate frm either 2.5 X 10 3 r 5 X 10 5 cells (nn-is mice nly) and 30 /x\ f a PCR master mix cnsisting f 50 mm KC1, 10 mm Tris-HCl, (ph 8.3), 1.5 mm MgCl 2, 1 nm f each primer, 200 nm dntps, 200 Mg/ml f gelatin, and 2.5 U f Taq plymerase (Perkin-Elmer Cetus, Nrwalk, CT). Amplificatins were perfrmed using a DNA thermal cycler (Perkin- Elmer Cetus, Nrwalk, CT) fr 40 cycles. Each cycle cnsisted f 94 C fr 75 secnds, 55 C fr 75 secnds, and 72 C fr 95 secnds, with a 2-secnd autextensin n the last segment fr each cycle. After amplificatin, 25 nl f the PCR prducts were electr-

3 1126 Investigative Ophthalmlgy & Visual Science, March 1994, Vl. 35, N. 3 phresed n 1.4% agarse gels in TBE electrphresis buffer (89 mm Tris-base, 89 mm bric acid, 2 mm EDTA, ph 8.0), transferred t a Hybnd+ filter membrane (Amersham Crp., Arlingtn Heights, IL) by the alkaline methd, 11 and hybridized with labeled prbe. The MCMV prbe was 3'-tailed with Digxigenin- 11-dUTP using terminal dexynucletidyl transferase and detected as specified by the manufacturer (Behringer Mannheim Crp., Indianaplis, IN). Experimental Plan The extent f T cell depletin in methylprednislne (sterid)-treated mice was determined 9 days after beginning immunsuppressin by sacrificing fur sterid-treated, MCMV-infected mice and fur uninfected, sterid-treated mice. Fur uninfected untreated mice were used as cntrls. Whle spleens were remved and strained thrugh nyln mesh t btain a single-cell suspensin; red bld cells were lysed by treatment with NH 4 C1. The cells were resuspended in serum-free tissue culture medium and cunted. The cells were stained with flurescein isthicyanate-cnjugated rat mnclnal antibdies specific fr muse CD3 (GIBCO BRL, Gaithersburg, MD), muse CD4 (GIBCO BRL), r muse CD8 (Bectn Dickinsn, San Jse, CA). Befre analysis, the stained cells were washed nce with PBS cntaining 50 tig/ml prpidium idide. 12 Flw cytmetry was dne using a Bectn Dickinsn FACStar PLUS flurescence-activated cell srter (Bectn Dickinsn). As shwn in Table 1, 93.0% t 93.8% f T cells were depleted in sterid-treated, MCMV-infected mice, and sterid treatment did nt preferentially deplete either CD4 + r CD8 + T cells. In these studies, the left eyes f nn-is and IS BALB/c mice were injected with 5 X 10 2 PFU f MCMV via the supraciliary rute. Fr virus recvery frm the anterir segment and psterir segment f the eye, five IS mice and five nn-is mice were sacrificed n days 3, 6, 9, 12, and 15 (after inculatin). The injected eye was enucleated, and the cnjunctiva and extracular muscles were remved. The eyes were separated int an anterir segment and a psterir segment by cutting the glbe alng the utside f ra serrata with a micrsurgical knife and Vannas scissrs (Edward Week Inc., Research Triangle Park, NC). Fr virus recvery frm unseparated eyes and frm nncular tissues (salivary glands, lung, spleen, kidney, brain), three r fur MCMV-infected mice frm each grup were sacrificed n days 3, 6, 9, 12, 16, 20, 24, and 28 PI After their remval, all the cular and nncular tissues were hmgenized in 1.0 ml f serumfree tissue culture medium using a hand-held tissue hmgenizer (Bispec Prducts, Inc., Racine, WI). Bld was cllected frm each muse fr separatin f PBL. The titer f virus in the tissues and PBL was determined by plaque assay and expressed as PFU/ml f tissue hmgenate. PCR was used t determine if MCMV DNA was present in PBL. Results frm virus recvery studies were analyzed fr significance (P < 0.05 r better) using the Kruskal-Wallis test, and PCR results were analyzed by x 2 - RESULTS Virus Recvery Frm Ocular Tissues f IS and Nn-IS Mice Because nly IS mice develp necrtizing retinitis after supraciliary inculatin f 5 X 10 2 PFU f MCMV, and cytmegalic cells indicative f virus infectin are bserved in bth the iris and ciliary bdy in the anterir segment and thrughut the retina in the psterir segment f IS mice, 5 we hypthesized that immunsuppressin permits increased replicatin f virus in the injected eye. As shwn in Figure 1, virus replicatin was significantly increased and clearance f virus was delayed in the inculated eye f IS mice. Additinal virus recvery studies were perfrmed t TABLE l. T Cell Depletin in Sterid-treated BALB/c Mice Grup Ttal T Cells/Spleen* (% depletin)^ CD4 + T Cells/Spleen (% depletin) CD8 + T Cells/Spleen (% depletin) Cntrl Sterid]: Sterid + MCMV 3.89 X 10 7 (0.0) 5.51 X 10 5 (98.6) 2.65 X 10 6 (93.2) 2.41 X 10 7 (0.0) 3.54 X 10 5 (98.5) 1.69 X 10 6 (93.0) 1.56 X 10 7 (0.0) 1.97 X 10 5 (98.7) 9.75 X 10 s (93.8) * Average number frm fur mice in each grup. t % depletin = 1 (mean cell number frm sterid-treated mice/mean cell number frm cntrl mice) X 100. X 2.0 mg methylprednislne given 3 times 4 days apart; mice were killed 1 day after the final injectin. 2.0 mg f methylprednislne given n day 2, +2, and +6; 5 X 10' 2 pfu MCMV injected n day 0 via the supraciliary rute. Mice were killed n day 7 pi.

4 MCMV Infectin in Immunsuppressed Mice r Q_ ct 2 O Q_ 1.5 O FIGURE l. Recvery f virus frm the injected eye f IS (O-O) and nn-is ( - ) BALB/c mice after inculatin f 5 X 10 2 PFU f MCMV via the supraciliary rute. Fur mice frm each grup were sacrificed at each time pint, and the amunt f virus in each specimen was determined (in duplicate) by plaque assay n mnlayers f MEF cells. The amunt f virus at each time pint is the mean virus titer (expressed as Lg 10 PFU/ml ± SEM) f all samples harvested at that time. The dashed hrizntal line indicates the lwer limit f virus detectin. *Significantly different frm nnimmunsuppressed grup (P < 0.05 r better) O IS Nn-IS 18 determine whether higher titers f virus were recvered frm the anterir and/r psterir segment f the inculated eyes f IS mice than frm the inculated eyes f nn-is mice. Significantly higher titers f virus were recvered at days 9, 12, and 15 PI in the anterir segment (Fig. 2A) and at day 15 PI in the psterir segment (Fig. 2B). In the psterir segment, the differences between the IS grup and the nn-is grup apprached significance at days 9 and 12 PI with P values f and 0.054, respectively. Virus Recvery Frm Nncular Tissues f IS and nn-is Mice We als determined the titer f virus in several nncular tissues (salivary glands, lungs, spleen, kidneys, brain) f IS and nn-is mice after supraciliary inculatin. In general, the titer f virus in IS mice was higher in all sites and clearance f virus was delayed cmpared t nn-is mice. In IS mice, significantly higher titers f virus (P < 0.05 r better) were recvered frm the salivary glands beginning at day 9 PI (Fig. 3A), and frm the lungs by day 12 PI (Fig. 3B). There was n significant difference in the amunt f virus recvered frm the spleens f IS mice cmpared with nn-is CL 1-5 O 1.0 B FIGURE 2. Recvery f virus frm the (A) anterir segment and (B) psterir segment f the injected eye f IS (O - O) and nn-is ( - ) BALB/c mice after inculatin f 5 X 10 2 PFU f MCMV via the supraciliary rute. Five mice frm each grup were sacrificed at each time pint, and the amunt f virus in each specimen was determined (in duplicate) by plaque assay n mnlayers f MEF cells. The amunt f virus at each time pint is the mean virus titer (expressed as lg 10 PFU/ml ± SEM) f all samples harvested at that time. The dashed hrizntal line indicates the lwer limit f virus detectin. *Significantly different frm nnimmunsuppressed grup (P < 0.05 r better). 18

5 1128 Investigative Ophthalmlgy & Visual Science, March 1994, Vl. 35, N. 3 ID CL A B O IS Nn-IS Q_ O FIGURE 3. Recvery f virus frm the (A) salivary glands, (B) lungs, and (C) spleen f IS (O-O) and nn-is ( - ) BALB/c mice after inculatin f 5 X 10 2 PFU f MCMV via the supraciliary rute. Fur mice frm each grup were sacrificed at each time pint, and the amunt f virus in each specimen was determined (in duplicate) by plaque assay n mnlayers f MEF cells. The amunt f virus at each time pint is the mean virus titer (expressed as lgj 0 PFU/ml ± SEM) f all samples harvested at that time. The dashed hrizntal line indicates the lwer limit f virus detectin. *Significantly different frm nnimmunsuppressed grup (P < 0.05 r better) mice at any time after infectin (Fig. 3C). Althugh n virus was recvered frm the kidneys f nn-is mice at any time after infectin, at later time pints (days 24 and 28 PI), 3.2 t 3.5 lgi 0 PFU/ml were recvered frm the kidneys f IS mice (data nt shwn). N infectius virus was recvered frm the uninculated eyes, brains, and PBL f either IS r nn-is mice at any time after infectin (data nt shwn). PCR Detectin f MCMV in Muse Peripheral Bld Leukcytes After inculatin f MCMV via the supraciliary rute, virus recvery studies indicated that MCMV spread t nncular tissues f IS and nn-is mice within a few days. These results suggested that virus spread frm the inculated eye t extracular sites hematgenusly, as has been demnstrated after inculatin f this virus via nncular rutes; 13 " 15 hwever, n virus was detected in the PBL by plaque assays. T determine whether virus was spread hematgenusly in amunts t lw t be detected by plaque assay, PCR was used t amplify MCMV sequences in DNA extracted frm PBL (Fig. 4). All the DNA samples frm mice were psitive fr MLTR. As Table 2 demnstrates, when the DNA frm 2.5 X 10 3 PBL frm IS mice was amplified fr MCMV DNA sequences, 67.6%

6 1129 MCMV Infectin in Immunsuppressed Mice < 400 bp FIGURE 4. Representative Suthern hybridizatin f PCR amplificatin prducts using MCMV-specihc amplimers and DNA lysates f peripheral bld leukcytes frm imnuinsuppressed (IS) r nn-immunsuppressed (nn-is) BA1.B/ c mice injected with 5 X 10" PKU f MCMV via the supraciliary rute. Clells were cllected and the DNA was extracted, amplified, and hybridized as described in Materials and Methds. Lanes: i and 20, <1 kb ladder; 2 t 5, IS day 0 PI; 9 t 12. IS day 24 PI; 6 t 8, nn-is day 9 PI: 13 t 15, nn-is day 24 PI; 16, cular tissue, uninfected BAI.B/c muse; 17, injected eye, MCMV-infected BALB/c muse; 18, MMTV-transfrmed muse cells; 19, nrmal human peripheral bld leukcytes. (23/34) f the samples were psitive fr MCMV. When the DNA frm the same number f PBL frm nn-is mice was amplified fr MCMV sequences, nly 3.8% (1/26) f the samples were psitive fr MCMV DNA. When the DNA frm 200-fld mre cells f nn-is mice was amplified fr MCMV, 58.3% (14/24) f the samples were psitive. The plasma and the serum f bth IS and nn-is mice were negative fr virus sequences (data nt.shwn), prviding further supprt fr the idea that virus was disseminated frm the inculated eye via leukcytes after supraciliary inculatin. MCMV replicated in bth grups f mice until day 6 PI. After this time, the titer f virus in nn-is mice decreased, whereas the amunt f virus in the injected eyes f IS mice remained significantly elevated. Virus des nt replicate in the retina f nn-is mice (as demnstrated micrscpically by lack f cytmegalic cells).5 Because there was essentially n virus replicatin in the psterir segment f nn-is mice by virus recvery frm separated psterir segments in these studies, it is likely that the majrity f the replicating virus recvered frm the unseparated inculated eyes f nn-is mice reflects virus replicatin in the anterir segment. Because significant virus replicatin ccurred in the psterir segment f IS mice, it appears that an intact immune system prevents spread f virus t and subsequent virus replicatin in the psterir segment after supraciliary inculatin. When virus is injected via the supraciliary rute, the site f injectin is juxtapsed t the anterir part f the uveal tract (i.e.. ciliary bdy), and cells f the uveal tract are permissive fr MCMV infectin.16"18 The results f these studies, therefre, suggest that ne rute by which MCMV spreads t the psterir segment f the injected eye is via the cells f the uveal tract. Because virus replicatin in the psterir segment was significantly higher in IS mice and since virus-infected cytmegalic cells have been bserved in the retina after supraciliary inculatin f MCMV,5 the results f the studies presented in this cmmunicatin prvide further supprt DISCUSSION After supraciliary inculatin f 5 X 102 PFU f MCMV: (1) significantly higher titers f virus were recvered frm the anterir segment and psterir segment f inculated eyes, salivary glands and lungs f IS mice than frm the same sites f nn-is mice; (2) the frequency f MCMV-psitive cells in peripheral bld leukcytes frm IS mice was apprximately 200-fld greater than frm nn-is mice; and (3) clearance f virus frm the inculated eye and nncular tissues was delayed in IS mice. These results als demnstrate that retinitis in IS mice crrelates with replicatin f virus in the psterir segment. Althugh the virus titratin results revealed that virus replicated in the anterir segment f bth IS and nn-is mice, the titer f virus in the anterir segment was significantly higher in IS mice. Because previus studies have demnstrated that the epithelium f the ciliary bdy and the epithelium f the iris are infected with virus and eventually becme necrtic in IS mice,5 the results f these experiments supprt the cnclusin that the anterir segment destructin bserved in IS mice results frm replicatin f virus. Cmparisn f the time curse f virus recvery frm unseparated eyes f bth IS and nn-is mice (Fig. 3A) revealed that 2. Amplificatin f MCMV DNA in Peripheral Bld Leukcytes frm IS and nn-is Mice TABLE 2.5 X Day PI Ttal Sterui w* Xne 1/2+ 0/2 2/4 3/4 4/4 3/4 3/4 3/4 2/4 2/4 23/34 1/3 1 /26 5X Sterid NO: NI) NI) W* Nne 3/3 1,/3 14/24" * Peripheral bld leukcytes were islated n I.vmphlyte-M gradients and amplified lr MCMV sequences as described in Materials and Methds. t Number f mice psitive lr MCMV sequences ttal. X = nt determined due t lw numbers f cells recvered frm IS mice. Significantly different frm cntrl mice ('J.5 X 10' cells). / ' < N significant difference between sterid-treated mice ('2.5 X 10:( cells) and cntrl mice (5 X 10'' cells).

7 1130 Investigative Ophthalmlgy 8e Visual Science, March 1994, Vl. 35, N. 3 fr the hypthesis that the acute retinitis that prgresses t retinal destructin in IS mice reflects replicatin f virus in bth the uveal tract and the retina. Several nncular sites were infected with virus after supraciliary infectin. PCR amplificatin f DNA frm PBL f IS mice demnstrated that PBL were virus-psitive by day 1 PI, and verall, 67.6% f the samples frm IS mice cntained viral DNA. Thus, leukcytes are infected early in the curse f MCMV infectiri7and hematgenus seeding prbably serves as majr rute f virus spread after supraciliary inculatin as it des after inculatin by ther rutes. 13 " 15 We were unable t detect MCMV in circulating leukcytes by plaque assay. The failure t recver virus frm PBL cntrasts with the results f Bale and cwrkers wh islated virus frm circulating leukcytes after intracular inculatin f 3 X 10 4 PFU f MCMV in IS mice 19 and als with the results f Bale and O'Neil wh recvered virus and viral DNA frm circulating leukcytes after intraperitneal injectin f 10 5 PFU f MCMV. 13 The difference in virus recvery frm leukcytes between ur studies and thse f thers may reflect the lwer dse f virus used fr the supraciliary injectin r the fact that nly a small fractin f a particular ppulatin f leukcytes appears t be infected with MCMV. 15>20>21 Additinally, amplificatin f DNA sequences des nt prvide infrmatin abut the status f the virus, and further studies will be needed t determine whether the virus carried by PBL after supraciliary inculatin is capable f prductive infectin. Althugh ther rgans were infected with virus after supraciliary inculatin, virus did nt spread t the brain r t the uninculated eye. These results cnfirm that virus spread t nncular tissues after supraciliary inculatin was nt via neural pathways (unlike herpes simplex viruses) and suggest that MCMV infectin resulting frm unicular supraciliary injectin cmprmises nly the bld-cular barrier f the injected eye. By extraplatin t CMV retinitis in human patients with AIDS, these results suggest that breakage f the bld-cular barrier may cntribute t the establishment f cular CMV infectin and/ r that cular CMV infectin requires a cmprmised bld-cular barrier. The ability f MCMV t spread t varius distant rgans was unrelated t the immune status f the muse. Althugh virus recvery studies demnstrated that virus spread initially t the same sites in IS and nn-is mice, virus replicatin was increased and clearance was prlnged amng nncular tissues (salivary gland, lung, and kidney) nly in IS mice. In IS mice, significant titers f virus were still recvered as late as day 28 PI, when virus had been cleared frm mst sites in nn-is mice. The bservatin that extensive tissue replicatin f virus ccurred in IS mice is in agreement with previus investigatins demnstrating increased virus replicatin and/r disease in IS animals. 19l22 ~ 28 Facilitatin f virus replicatin and the delayed clearance f virus after supraciliary inculatin f MCMV in IS mice mst likely reflect the prfund immunsuppressin induced by treatment with methylprednislne that affects bth specific and nnspecific immune effectr functins. Fr example, sterid treatment wuld reduce r eliminate CD4 + T cells required fr clearance f virus frm the salivary gland and CD8 + T cells, which are needed fr clearance f MCMV frm nnsalivary gland sites and fr prtectin frm MCMV retinitis In cnclusin, the results f these studies cmparing IS and nn-is mice after supraciliary inculatin f MCMV demnstrated that sterid-induced immunsuppressin enhances virus spread (frm the anterir segment t the psterir segment) and virus replicatin in the inculated eye, increases virus replicatin significantly in nncular tissues, and delays clearance f virus frm infected cular and nncular tissues. Studies are in prgress t elucidate the mechanisms by which hematgenus disseminatin f cell-assciated MCMV results in extracular infectin and replicatin after supraciliary inculatin f IS BALB/c mice. Key Wrds BALB/c muse, retinitis, murine cytmegalvirus, supraciliary inculatin, immunsuppressin Acknwledgments The authrs thank Drs. Sctt W. Cusins, Cecelia Cruse, and Vincent R. Vann fr prviding helpful discussins, technical assistance, and materials. The authrs als thank Dr. Atsushi Azumi and Mr. Charles Thmas fr assistance with flw cytmetry and Ms. Jyce Schiffman (Department f Ophthalmlgy, University f Miami Schl f Medicine) and Dr. Susan Hilsenbeck (Department f Medicine, University f Texas Health Science Center) fr assistance with statistical analyses f the virus recvery results and f the PCR studies. References 1. Blm JN, Palestine AG. The diagnsis f cytmegalvirus retinitis. Ann Intern Med. 1988; 109: Drew WL. Cytmegalvirus infectin in patients with AIDS. Clin Infect Dis. 1992; 14: Pepse JS, Hlland GN, Nestr MS, Cchran AJ, Fs RY. Acquired immune deficiency syndrme: Pathgenic mechanisms f cular disease. Ophthalmlgy. 1985; 92: Athertn SS, Newell CK, Kanter MY, Cusins SW. Retinitis in euthymic mice fllwing inculatin f murine cytmegalvirus (MCMV) via the supraciliary rute. Curr Eye Res. 1991; 10: Athertn SS, Newell CK, Kanter MY, Cusins SW. T cell depletin increases susceptibility t MCMV retinitis. Invest Ophthalml Vis Sci. 1992;33: Saiki RK, Scharf S, Falna F, et al. Enzymatic ampli-

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