Published online before print October 20, 2004
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Comparative and Experimental Medicine, College of Veterinary Medicine, University of Tennessee, Knoxville
1 Correspondence: M409 Walters Life Sciences Bldg., University of Tennessee, Knoxville, TN 37996-0845. E-mail: btr{at}utk.edu
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Key Words: immunopathology • Herpes virus • cornea • T lymphocyte
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Recently, the Carbone laboratory [6 ] developed transgenic mice using the T cell receptor (TCR) of B6 mice, which recognize the immunodominant H-2Kb-restricted peptide SSIEFARL (gB498–505). In such mice, >98% of the CD8+ T cells recognize SSIEFARL-sensitized target cells [6 ]. Such animals generate SSIEFARL-specific effector T cells rapidly after virus infection, and such cells are highly effective at recognizing HSV-infected target cells in vitro [6 ]. As the CD8+ SSIEFARL response has been shown to be protective, such animals were suspected to be highly resistant to HSV and perhaps not succumb to ocular lesions. Moreover, as the frequency of effector T cells to HSV was extremely high, it was anticipated that it should be readily possible to demonstrate cells of this reaction in the eye if the HSV-specific CD8+ response was involved in the immunopathology.
It is surprising, however, that as documented in this report, whereas gBT mice do indeed succumb to HSK with a mean lesion score marginally less than B6 controls, their ocular lesions have few if any SSIEFARL-specific T cells. Indeed, these HSK lesions, as in B6 animals, are dominated by CD4+ T cells and are the only cells demonstrable in the later phases of the disease. Reasons why such HSV-specific CD8+ T cells fail to enter the eye, whereas they do so efficiently in the innervating trigeminal ganglion (TG), are discussed.
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5% and 22% of total white blood cells (WBCs), respectively, compared with 12% of CD4+ and 7% of CD8+ T cells of the C57BL/6 mice peripheral blood WBCs. C57BL/6 (B6) mice (CD45.2+) were purchased from Harlan-Sprague-Dawley (Indianapolis, IN). Recombination-activating gene (RAG)–/– mice (B6.129S7-Rag1tm1Mom/J), 6–8 weeks of age, were purchased from the Jackson Laboratory (Bar Harbor, ME). Animals were age- and sex-matched for all experiments. All manipulations involving immunocompromised mice were performed in laminar flow hood. To prevent bacterial superinfections, mice received prophylactic treatment, starting 1 day prior to corneal infection, with Sulfatrim pediatric suspension (Barre National, Baltimore, MD) at the rate of 5 ml per 200 ml drinking water. All experimental procedures were in complete agreement with the Association of Research in Vision and Ophthalmology (Rockville, MD) resolution about the use of animals in research.
Virus
HSV-1 RE "Hendricks" (obtained from the laboratory of Dr. Robert Hendricks when at the University of Illinois, Chicago), "Tumpey" (obtained from Terence Tumpey when at the University of South Alabama, Mobile), and KOS strains were propagated and titrated on monolayers of Vero cells (American Type Culture Collection, Manassas, VA, Cat. No. CCL81) using standard protocols [7
]. HSV-1 RE isolates were used for corneal infections, and HSV-1 KOS was used to immunize mice.
Corneal HSV infections and clinical observation
Corneal infections of all mice groups were conducted under deep anesthesia induced by avertin (Sigma Chemical Co., St. Louis, MO). B6 and gBT mice were ocularly infected with the Tumpey isolate of HSV-1 RE, and RAG–/– ocular infections were carried out with the Hendricks isolate. Mice were scarified on their corneas with a 27-gauge needle, and a 4-µl drop containing 5 x 106 plaque-forming units (pfu) of the respective HSV-1 RE isolate was applied to the eye and gently massaged with the eyelids. The eyes were examined on different days p.i. with a slit lamp biomicroscope (Kowa, Nagoya, Japan), and the clinical severity of keratitis of individually scored mice was recorded. The scoring system was as follows: +1, mild corneal haze; +2, moderate corneal opacity or scarring; +3, severe corneal opacity, but iris-invisible; +4, opaque cornea; and +5, necrotizing stromal keratitis.
Detection and titration of replicating virus from corneal swabs
Eye swabs were taken from infected corneas (four eyes/group) using sterile swabs soaked in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 IU/ml penicillin and 100 µg/ml streptomycin. Swabs were put in sterile tubes containing DMEM and stored at –80°C. For detection of virus, samples were thawed and vortexed. Duplicate 200 µl aliquots of dilutions of each sample were plated on Vero cells grown to confluence in 24-well plates at 37°C in 5% CO2 for 11/2 h. Medium was aspirated, and 500 µl 2x DMEM containing 1% low melting-point agarose was added to each well. Titers were calculated as log10 pfu/ml as per standard protocol [7
].
Adoptive transfer of HSV-immune CD4+ and CD8+ T cells
To generate virus-activated CD4+ and CD8+ T cells, B6 and gBT mice were injected with 5 x 106 pfu HSV-1 KOS into the footpad. Single-cell suspensions of pooled spleens and popliteal lymph nodes were prepared from immunized mice 7–8 days later, and the T cell subsets were purified using a mouse CD4 or CD8 subset column (R&D Systems, Minneapolis, MN). By flow cytometry analysis, the purified populations consisted of 80% CD4+ or CD8+ T cells. Approximately 50% of the CD8+ T cells from the gBT mice (compared with only 15% of splenic CD8+ T cells from unmanipulated mice) produced interferon-
(IFN-) upon in vitro restimulation to the gB498–505 immunodominant epitope of HSV, measured by intracellular IFN- production [measured prior to purification from cell samples taken from pooled splenocyte and draining lymph node (DLN) single-cell suspensions; see below for details]. CD4+ T cells from virus-immunized mice showed the activated phenotype as judged by flow cytometry analysis using the CD25, CD44hi, and CD69 cell-surface activation markers compared with CD4+ T cells from naïve B6 mice (not shown). In cotransfer experiments, RAG–/– mice received activated CD4+ and CD8+ T cells, each at 2 and 3 days postocular infection, respectively [5x106 purified cells each subset intravenously (i.v.)]. In single-cell transfer experiments, RAG–/– mice received 107-purified CD4+ or CD8+ cells i.v. at days 2 and 3 postocular infection, respectively.
Cellular analysis and flow cytometry
Surface staining for the detection of adoptively transferred T cells
Single-cell suspensions were prepared from spleen and DLN of mice. The DLNs that were used for cellular analysis were the mandibular and the superficial cervical lymph nodes. Isolated corneas and TGs were pooled, minced, and digested in 1 mg/ml collagenase/dispase (Roche Diagnostics, Mannheim, Germany) for 11/2 h followed by two washes in phosphate-buffered saline (PBS). Surface staining of cells was carried out in flow cytometry buffer (1x PBS with 3% fetal calf serum and 0.1% sodium azide). Viable cells (106) were blocked with Fc block (Clone 2.4 G2, PharMingen, San Diego, CA). Cells from spleens, DLNs, TGs, or corneas fluorescein isothiocyanate (FITC)-labeled anti-CD4 (RM4-5, PharMingen) and anti-CD8 (Clone 53-6.7, PharMingen) monoclonal antibodes (mAb) were used. Events were collected on FACScan (Becton Dickinson, San Jose, CA) and analyzed using Cellquest Version 3.0 (Becton Dickinson).
Quantification of IFN- production by intracellular staining
To enumerate IFN--producing CD8+ T cells, intracellular cytokine staining was performed as described previously [8
]. In brief, 106 splenocytes or DLN cells were cultured in flat-bottom 96-well plates. Cells were left untreated, stimulated with HSV gB498–505 peptide (SSIEFARL) and ovalbumin257–264 peptide (SIINFEKL; 1 µg/106 cells), or treated with phorbol 12-myristate 13-acetate (10 ng/ml) and ionomycin (500 ng/ml) and were incubated for 6 h at 37°C in 5% CO2. Brefeldin A (10 µg/ml) and interleukin-2 (50 U/ml) were added for the duration of the culture period. After this period, cell-surface staining was performed as described above. This was followed by intracellular cytokine staining using a cytofix/cytoperm kit (PharMingen) in accordance with the manufacturer’s recommendations. Phycoerythrin-labeled anti-IFN- antibody was used for intracellular cytokine staining (Clone XMG1.2, PharMingen). Events were collected on FACScan (Becton Dickinson) and analyzed using Cellquest Version 3.0 (Becton Dickinson).
Histopathology, immunohistochemistry, and immunofluorescence
Eyes were enucleated and fixed in 10% buffered neutral formalin and embedded in paraffin. Sections (5 µm thick) were cut, deparaffinized, and stained with haematoxylin and eosin (H&E).
For immunofluorescence analysis, eyes were enucleated at indicated time-points, frozen in optimum cutting temperature compound (Miles, Elkart, IN). Sections (6 µ thick) were cut, air dried, and fixed in acetone:methanol (1:1) at –20°C for 10 min. Endogenous biotin was blocked in acetone:methanol fixed frozen sections with an endogenous biotin-blocking kit (Molecular Probes, Eugene, OR). This was followed by blocking with 5% bovine-serum albumin (BSA)-PBS–0.05% Tween 20 containing 1/200 dilution of Fc block (Clone 2.4G2, PharMingen) for 2 h. Antibodies used for staining were anti-CD4 and anti-CD8-FITC (5 µg/ml, PharMingen). Antibody dilutions were made in 1% BSA-PBS. After incubating overnight at 4°C, slides were washed thoroughly in PBS. For slides stained with FITC-labeled antibodies, propidium iodide (PI) was used as a counterstain (Vectashield with PI, Vector Laboratories, Burlingame, CA). Images were captured with a Leica SP2 laser-scanning confocal microscope.
Statistical analysis
Wherever specified, data obtained were analyzed for statistical significance by a one-tailed standard Student’s t-test.
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3, and the incidence was only marginally lower than that seen in the B6 animals (63%; Fig. 1B
). However, mean lesion scores between the two strains were comparable (Fig. 1A) . Lesions were typically manifest as corneal opacity, necrosis, ulceration, and growth of blood vessels into the normally avascular cornea (neovascularization; Fig. 1C
). Eyes with scores greater than 3 were processed for histopathological examination, which revealed inflammatory changes in the corneal epithelial and the stromal layers and the infiltration of a large number of inflammatory cells (Fig. 1C)
.
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Figure 1. Development of HSK in the gBT mouse. (A) Kinetics of SK development in the two strains of mice after ocular infection with HSV-1 RE Tumpey isolate. Data represent the mean lesion score ± SD (n=8) at different days p.i. (B) Lesion scores at day 15 p.i. Each symbol represents the lesion score for an individual eye for the group. Horizontal bars and numbers in parentheses represent the mean lesion score for the particular group. Percentages refer to the incidence of lesions with scores 3. (C) Representative photographs of eyes with SK lesions with opacity and angiogenesis at day 15 p.i. (left panels). The same eyes were processed, embedded in paraffin, and stained with H&E for histological examination (right panels). (D) Photographs of the periocular area of infected B6 and gBT mice at day 9 p.i., showing the presence (B6) or the absence (gBT) of facial lesions.
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Corneal viral replication in gBT mice
There was no difference in virus replication in corneas of gBT versus control B6 mice. As measured by corneal swabs at days 3 and 5 p.i., the number of recoverable virions was not significantly different between the two strains of mice (Fig. 2A
). In addition, as with the control mice, replicating virus was no longer demonstrable by day 7 p.i. in gBT mouse corneas (Fig. 2A)
. Thus, a difference in virus replication was not an explanation for the HSK susceptibility of gBT mice.
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Figure 2. Ocular viral clearance and infiltration of CD4+ and CD8+ T cells into infected corneas of B6 and gBT mice. (A) Detection and quantification of replicating virus by ocular swabs at days 3, 5, and 7 p.i. Data represent mean ± SD (n=4). (B) Frozen sections (6 µ) of gBT and B6 eyes at day 7 p.i. and with SK lesions 3 at day 15 p.i. were processed for staining with FITC-labeled anti-CD4 and anti-CD8 mAb (PharMingen). Photographs show cells in the peripheral cornea at day 7 p.i. (the origin of the iris is shown by an open arrow wherever possible) and central cornea at day 15 p.i. Solid arrows show CD4+ and CD8+ T cells. Counterstaining was done with PI. Original bar, 80 µm.
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3) to determine the phenotype of the infiltrating T cells. As visualized by immunohistochemistry, CD4+ T cell infiltrates were seen in the stroma of infected B6 eyes in the peripheral cornea (limbus and paracentral cornea) as early as day 7 p.i. (Fig. 2B)
. At day 15 p.i., these cells had reached the central cornea (Fig. 2B)
and represented 2.5% of the total corneal cells, as measured by flow cytometry (Table 1
). CD8+ T cell infiltrates, conversely, although demonstrable in the peripheral cornea at day 7 p.i. (Fig. 2C)
, were scarcely demonstrable at day 15 p.i. in the peripheral or central corneal regions (Fig. 2C)
, representing only 0.2% of the total corneal cells (Table 1)
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Table 1. CD4+ but Not HSV-Reactive CD8+ T Cells Are Demonstrable in SK Lesions
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CD4+ but not HSV-reactive CD8+ T cells aid corneal lesions
HSV-reactive CD8+ T cells facilitate corneal viral clearance
To analyze further the role of the T cell subsets in corneal lesions, HSV-activated CD4+ (from B6) and CD8+ (from gBT mice) were adoptively transferred into T and B cell-deficient, ocular HSV-1 Hendricks-infected RAG–/– mice. These experiments were designed to further confirm the inability of the HSV-reactive CD8+ but not the CD4+ T cells to migrate to corneal lesions. RAG–/– mice received activated CD4+ and CD8+ T cells in cotransfer experiments using a transfer protocol (see Materials and Methods), which allows mice to survive beyond day 12 p.i. and ensure optimal HSK development in surviving mice. With this adoptive transfer protocol, 60% of mice survived until day 15 p.i. (the last time-point analyzed; Fig. 3A
) with an HSK incidence (lesion score 3) of 67% (Fig. 3B)
. Flow cytometry analysis of the DLNs and the spleens of these mice revealed that CD8+ T cells outnumbered CD4+ T cells almost two- and sixfold in the respective organs (of the total cells in each organ tested, in DLNs, CD8 comprised 1.3±0.5%, and CD4 was 0.9±0.4%; in spleens, they were 5.5±0.8% and 0.6±0.3%, respectively). Despite this, however, and in line with results described above, in chronic lesions, CD4+ T cells but not CD8+ T cells were present, as judged by frozen-section immunohistochemistry (Fig. 3C)
.
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Figure 3. Adoptively transferred CD4+ T cells are present in corneal lesions of RAG–/– mice. Graph shows the survival of RAG–/– mice reconstituted with CD4+ or CD8+ or both after ocular infection with the HSV-1 RE Hendricks isolate (n=5 at the beginning of the study). (A) Lesion scores in ocularly infected RAG–/– mice receiving CD4+ and CD8+ T cells on the day mice were killed. Each symbol represents the lesion score for an individual eye for the group. Horizontal bars and numbers in parentheses represent the mean lesion score for the particular group. Percentages refer to the incidence of lesions with scores 3. (B) Frozen sections (6 µ) of eyes of RAG–/– mice receiving T cell adoptive transfers with lesion scores 3 (for RAG–/– mice receiving CD8+ T cells, eyes with scores of 1) were processed for staining with FITC-labeled anti-CD4 and anti-CD8 mAb (PharMingen). The origin of the iris is shown with an open arrow for the corneal photograph at day 7 p.i. Solid arrows show the location of the respective cells in the central corneal region. Counterstaining was done with PI. Original bar, 80 µm.
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3) were evident in such animals at the time of sacrifice (incidence of 63%, Fig. 3B
). Adoptively transferred CD4+ T cells could be recovered readily from spleens and DLNs (of the total cells in each organ, 9±0.4% in the spleens and 5±1.7% in the DLNs), and a larger number of activated transferees were in the DLNs (in the DLNs, 59±8.5% CD4+CD25+, 19±2% CD4+CD62L+, and 19±5% CD4+CD69+; in the spleens, 18±4.6% CD4+CD25+, 3±0.5% CD4+CD62L+, and 4.2±0.3% CD4+CD69+). These cells were also demonstrable in corneas, showing severe lesions by immunohistochemistry (Fig. 3C) . In contrast, mice receiving only HSV-reactive CD8+ T cells were protected from encephalitis and survived beyond day 12 p.i. (Fig. 3A)
. In such mice, as with the infected B6 and gBT corneas, CD8+ T cells were evident in the peripheral cornea at day 7 p.i. by immunohistochemistry (Fig. 3C)
. Other animals of the group, terminated at day 15 p.i., developed only mild lesions (0% incidence of HSK lesions with scores 3), unlike their CD4+-reconstituted counterparts (Fig. 3B)
. At day 15 p.i., adoptively transferred IFN--producing CD8 was readily recoverable from spleens and DLNs. [Cells (5.9±1.7%) in the spleen and 5±0.9% cells in the DLNs of the total cells were CD8+ T cells. Of these recovered CD8+ T cells, 40–50% were IFN- producers when stimulated in vitro with the immunodominant gB peptide-epitope.] However, such cells were not demonstrable in eyes with mild lesions (Fig. 3C)
. Replicating virus was readily demonstrable from eyes of mice receiving CD4+ T cells at days 7 and 12 p.i., when they were killed (Fig. 4A ). In contrast, in mice that received CD8+ T cells only, virus was unrecoverable at day 7 p.i. in a majority of eyes, and at the time of sacrifice, replicating virus was not demonstrable in any of the eyes (Fig. 4A)
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Figure 4. Protection by HSV-reactive CD8+ T cells in the TG. (A) Clearance of corneal virus in RAG–/– mice receiving B6 CD4+ or gBT CD8+ T cells at the indicated time-points. Corneal swabs were used to measure titers. Each dot represents virus titer from one eye. (B) TGs from ocularly infected gBT mice and RAG–/– mice receiving T cell adoptive transfers were processed for frozen section (6 µ) immunohistochemistry at the indicated time-points using FITC-labeled anti-CD4 and anti-CD8 mAb (PharMingen). Arrows show the respective cells. Counterstaining was done with PI. Original bar, 80 µm.
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HSV-reactive CD8+ T cells in TGs provide additional protection
Further experiments were carried out to test the notion that the HSV-reactive CD8+ T cell, in addition to functioning in the corneal environment, may protect by its effect at the TG. gBT mice and RAG–/– mice receiving HSV-reactive CD8+ T cells were killed at days 7 and 15 p.i. These mice, as described above, clear ocular virus within 7 day p.i., survive encephalitis, and are protected from virus-induced facial lesions. In these mice, HSV-reactive CD8+ T cells were demonstrable by immunohistochemistry in the TGs at day 7 p.i. (Fig. 4B)
and although were not evident in corneal lesions at day 15 p.i., were readily demonstrable at this time-point in the TGs (Fig. 4B) . In mice that possessed CD4+ T cells in addition to the HSV-reactive CD8+ T cells (gBT and RAG–/– mice with cotransferred CD4+ and CD8+ T cells), CD4+ T cells were seen in the TG at days 7 (not shown) and 15 p.i. (Fig. 4B)
. It is interesting that although in the lymphoid organs of such mice, the numbers of CD4+ T cells were fewer than CD8+ T cells, they were almost equally represented in the TGs (Table 2
). The TGs from RAG–/– mice that were reconstituted with activated CD4+ T cells from B6 mice similarly also contained the adoptively transferred cells (not shown). Accordingly, the protective nature of the HSV-reactive CD8+ T cells may be a result of their ability to enter the TG and function as immune surveyors of viral replication, as described by others [10
11
12
13
14
], and prevent reactivation and spread to the cornea and the central nervous system.
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Table 2. CD4+ and HSV-Reactive CD8+ T Cells Are Demonstrable in the TG
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The apparent selective nature of the corneal inflammatory milieu that recruits the T cells based on subtype or antigen specificity is perplexing and at present, lacks a mechanistic explanation. It is also apparent from other studies that recognition of viral antigens is not a prerequisite for CD4+ or CD8+ T cells to migrate to HSK lesions [8 , 23 24 25 26 ]. However, with the immunocompetent mouse model, results suggest that nonspecific and virus-reactive CD4+ T cells infiltrate the cornea, the former albeit dominating the latter during the chronic phase [23 ]. The bottom line is that in any case, CD4+ T cell types are capable of infiltration. However, it is surprising that although the nonspecific CD8+ T cell readily migrates to the cornea and mediates HSK lesions (our unpublished results and ref. [8 ]), the HSV-reactive CD8+ T cell was unable to enter chronic lesions but not early corneal lesions or lesions in the peripheral nervous system (PNS; unpublished results and the present study and refs. [10 , 13 , 14 ]). Our ongoing investigations are directed at understanding whether differences in homing molecule expression on T cells provide an explanation for this phenomenon, and if so, what factors dictate such differences.
The early influx of HSV-reactive CD8+ T cells into virus-infected corneas, evident from gBT and RAG–/– adoptive transfer experiments, appears to be, at least in part, directed by the presence of the virus itself and affects viral clearance. The lack of difference in ocular viral clearance, between the gBT and the B6 mouse, and the similarity in the CD8+ T cell response during the early stages in both of these mice suggest virus-reactive cells to comprise a majority of CD8+ T cells in the B6 mouse as well. Analyzing the ocular CD8+ T cell response to viral immunodominant epitopes and antigens further elucidates the identity of the CD8+ T cells at various stages in the disease.
As is evident from the RAG–/– mouse adoptive transfer experiments, protection from HSK and lethal encephalitis occurs when HSV-reactive CD8+ T cells alone are present. This is in line with previous studies in immunocompetent mice, showing that removing CD8+ T cells worsens lesions and increases susceptibility to encephalitis [9 , 20 ]. The presence of CD4+ T cells will, however, inevitably lead to corneal disease, regardless of the presence or absence of HSV-reactive CD8+ T cells. Whether virus-reactive or nonreactive, the CD4+ T cells are unable to clear ocular virus, unlike their HSV-reactive CD8+ counterparts. So, although activation of the HSV-reactive CD8+ T cell achieves a protective effect, on the contrary, CD4+ T cell activation leads to enhanced tissue destruction. It is still unclear as to how CD4+ T cells dominate the T cell response in the cornea and mediate the corneal tissue injury. It is important to understand the basis of this preferential recruitment of CD4+ over the CD8+ T cells in the cornea, as if the balance can be tipped in favor of the HSV-reactive CD8+ T cells, HSK lesions could possibly be ameliorated.
The mechanism that the virus-reactive CD8+ T cells use to rid the cornea, or for that matter, the PNS or in the DLNs, of virus is another matter of interest. The possibilities include the use of antiviral-soluble factors generated by the activated cells locally in the cornea or in an extraocular site (or both) or direct cytolysis of virus-infected cells. However, based on previous observations by the Hendricks group [10
11
12
13
14
], the former mechanism of noncytolytic control seems to be operative. The cytokine IFN-, known to inhibit HSV replication and readily produced by the virus-reactive cells when stimulated with the cognate viral peptide, has been suggested previously [14
, 27
28
29
30
]. Experiments designed to clarify these mechanisms are being done using antisera to neutralize candidate molecules involved as well as adoptive transfers of donor CD8+ T cells from cytokine knockout animals.
We show that CD8+ T control of virus in lesions results from their activity in the PNS as well. Following corneal infection, virus rapidly gains access to nerve endings and passes by retrograde transport to the TGs [31 , 32 ]. Here, it may replicate in some cells and induce a florid inflammatory response, which after a time, is dominated by CD8+ T cells [10 , 13 , 14 , 33 ]. As demonstrated elegantly by Leib and co-workers [34 ], this is followed by an anterograde transport of virus from the TG to the periocular regions, an event believed to be necessary for facial lesions seen normally in infected wild-type mice. These lesions eventually heal, possibly as a result of the infiltration and antiviral effects of the CD8+ T cells in the TG. The lack of facial lesions in the gBT mice therefore clearly shows the effectiveness of the virus-reactive CD8+ T cells at controlling replication in the PNS. This is also corroborated by recent results from the Carbone lab [35 ], using the zosteriform infection model. It is tempting to speculate that these cells functioned in the TG to curtail replication and stop virus transporting to the corneal stroma. Further evaluations will use virus strains that are unable to reactivate in the TG and transport to tissues such as the eye.
In conclusion, our results indicate that the CD4+ T cells perpetuate the virus-induced immunopathology, irrespective of antigen specificity. CD8+ T cells, recognizing cognate viral antigens, conversely, are capable of controlling the inciting infection, thus providing protective effects. Whether these mechanisms apply universally to most microbe-induced immunopathologies and whether such can be harnessed to develop common therapeutic modalities remain to be seen.
Received September 2, 2004; revised September 21, 2004; accepted September 23, 2004.
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Interferon can prevent Herpes simplex virus type 1 reactivation from latency in sensory neurons J. Virol. 75,11178-11184 interferon in control of Herpes simplex virus type 1 reactivation J. Virol. 73,3418-3423
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