ABSTRACT
Like varicella-zoster virus (VZV), simian varicella virus (SVV) reactivates to produce zoster. In the present study, 5 rhesus macaques were inoculated intrabronchially with SVV, and 5 months later, 4 monkeys were immunosuppressed; 1 monkey was not immunosuppressed but was subjected to the stress of transportation. In 4 monkeys, a zoster rash developed 7 to 12 weeks after immunosuppression, and a rash also developed in the monkey that was not immunosuppressed. Analysis at 24 to 48 h after zoster revealed SVV antigen in the lung alveolar wall, in ganglionic neurons and nonneuronal cells, and in skin and in lymph nodes. In skin, SVV was found primarily in sweat glands. In lymph nodes, the SVV antigen colocalized mostly with macrophages, dendritic cells, and, to a lesser extent, T cells. The presence of SVV in lymph nodes, as verified by quantitative PCR detection of SVV DNA, might reflect the sequestration of virus by macrophages and dendritic cells in lymph nodes or the presentation of viral antigens to T cells to initiate an immune response against SVV, or both.
IMPORTANCE VZV causes varicella (chickenpox), becomes latent in ganglia, and reactivates to produce zoster and multiple other serious neurological disorders. SVV in nonhuman primates has proved to be a useful model in which the pathogenesis of the virus parallels the pathogenesis of VZV in humans. Here, we show that SVV antigens are present in sweat glands in skin and in macrophages and dendritic cells in lymph nodes after SVV reactivation in monkeys, raising the possibility that macrophages and dendritic cells in lymph nodes serve as antigen-presenting cells to activate T cell responses against SVV after reactivation.
INTRODUCTION
Primary varicella-zoster virus (VZV) infection produces chickenpox (varicella), after which the virus becomes latent in ganglionic neurons along the entire neuraxis. As VZV-specific T cell immunity declines with advancing age, VZV reactivates to produce zoster, which may be complicated by multiple neurological and ocular disorders. Simian varicella virus (SVV) infection of nonhuman primates has served as a good model with which to study the pathogenesis of VZV because of the pathological, immunological, and virological similarities of SVV to VZV (1, 2). VZV reactivation is increased in patients receiving chemotherapy or after X-irradiation (3); similarly, SVV reactivates in latently infected African green monkeys (AGM) and cynomolgus monkeys (CM) after immunosuppression and environmental stress (4). In rhesus macaques, intrabronchial inoculation with SVV produces varicella, followed by the establishment of latency (5) and virus reactivation after X-irradiation (6, 7). At the time of SVV reactivation in CM, expression of CXCL10 (a chemokine which recruits activated T cells and NK cells) correlates with transient T cell infiltration in ganglia (8). In the study described here, we determined if a combination of irradiation and treatment with prednisone and tacrolimus induces reactivation of SVV in latently infected rhesus macaques to study the distribution of reactivated SVV in ganglionic and nonganglionic tissues.
MATERIALS AND METHODS
Monkeys.
Seven SVV-seronegative rhesus macaques were housed in the Tulane National Primate Research Center in Covington, LA, and used in all experiments. The ages, sexes, and weights of the monkeys used in these experiments are presented in Table 1.
TABLE 1.
Age, sex, and weight of the Indian rhesus macaques used in this study
| Monkey no. | Age (yr) | Sexa | Wt (kg) |
|---|---|---|---|
| HB62 | 3.5 | M | 5.2 |
| HI83 | 3.2 | M | 5.0 |
| HF39 | 3.3 | M | 5.1 |
| HC44 | 3.4 | M | 5.6 |
| HA95 | 3.5 | M | 5.3 |
| II49 | 3.6 | F | 5.5 |
| IK10 | 3.6 | F | 5.5 |
| IE04 | 3.0 | M | 5.4 |
M, male; F, female.
Establishment of latent SVV infection.
SVV (a deltaherpesvirus strain) isolated from a naturally infected monkey (Erythrocebus patas) was propagated in Vero (African green monkey kidney) cells, and a virus stock was prepared as described previously (9). SVV-seronegative rhesus macaques (monkeys HB62, HI83, HF39, HC44, HA95, II49, and IK10) were inoculated intrabronchially with 104 PFU of wild-type SVV (monkeys HB62, HI83, HA95, II49, and IK10) or SVV expressing enhanced green fluorescent protein (SVV-EGFP) (monkeys HF39 and HC44) as described previously (4, 10). Earlier, we demonstrated that SVV-EGFP is pathogenic in African green monkeys and that SVV-EGFP is mildly attenuated compared to wild-type SVV (11, 12). All monkeys were monitored by physical exams every 2 or 3 days, and blood samples were collected either weekly or biweekly. Monkeys HB62, HI83, HC44, HA95, II49, and IK10 developed a mild varicella rash at 10 to 14 days postinoculation (dpi), while monkey HF39 did not develop a rash but seroconverted. Four months later, monkeys II49 and IK10 were euthanized and lymph nodes and ganglia were collected. All procedures were performed following appropriate guidelines and protocols approved by the Institutional Animal Care and Use Committee of the Tulane National Primate Research Center.
Immunosuppressive regimens.
All immunosuppressive treatments were performed as described previously (4). Five months after primary infection, monkeys HB62, HI83, HF39, and HC44 were transported by van (2-h round trip) from the Tulane National Primate Research Center in Covington, LA, to the Tulane University Cancer Center in New Orleans, LA, anesthetized, and exposed to a single dose of 200-cGy total body X-irradiation and then started on daily oral treatment with 500 μg (80 μg/kg of body weight/day) of tacrolimus (Prograf) and 5 mg (2 mg/kg/day) of prednisone until they were euthanized. Monkey HA95 was not irradiated or treated with drugs but was transported with the experimental animals as described above (Fig. 1). All animals were monitored again by physical exams every 2 or 3 days, and blood samples were collected weekly until SVV reactivation.
FIG 1.
Primary SVV infection and experimental reactivation in rhesus macaques. Seven Indian rhesus macaques (monkeys HB62, HI83, HF39, HC44, HA95, II49, and IK10) were inoculated intrabronchially with 104 PFU of SVV or SVV-EGFP. With one exception, all monkeys developed varicella rash, and all became viremic at 10 to 14 days postinoculation. Two months later, two monkeys (monkeys II49 and IK10) were euthanized for analysis of latently infected tissues. Five months later, four monkeys (monkeys HB62, HI83, HF39, and HC44) were exposed to X-irradiation and treated orally with tacrolimus and prednisone daily for the duration of the experiment. All four immunosuppressed monkeys developed a zoster rash at 7 to 12 weeks postimmunosuppression. The nonimmunosuppressed monkey (monkey HA95) developed a zoster rash 6.5 months after virus infection. All five monkeys were euthanized 24 to 48 h after the appearance of the rash, and multiple tissue samples were analyzed for SVV antigens. m, months; w, weeks.
Harvesting and processing of tissues.
At 7 to 12 weeks, examination of the monkeys revealed a rash in all irradiated monkeys, including monkey HA95, which was transported to the irradiation facility. They were all euthanized within 24 to 48 h after the appearance of zoster, and lymph nodes, skin, and ganglia were harvested and fixed as described below for immunological, virological, and pathological analyses. Areas of skin containing the zoster rash were punch biopsied while the monkeys were under anesthesia, fixed in 4% paraformaldehyde, and embedded in paraffin. Samples of lung and ganglionic tissue from each dermatome were harvested from each monkey, fixed in 4% paraformaldehyde, and embedded in paraffin. A portion of the lymph node tissue samples was snap-frozen for DNA extraction, and the other portion was fixed in 4% paraformaldehyde and embedded in paraffin.
Determination of anti-SVV antibody levels.
Anti-SVV antibody titers in serum obtained from all monkeys before and after varicella and at necropsy were determined using a plaque reduction assay as described previously (13). Briefly, dilutions of heat-inactivated sera from monkeys were mixed with an equal volume of Dulbecco modified Eagle medium containing 100 to 200 PFU of SVV. Serum-virus mixtures were incubated for 1 h at room temperature and plated onto a monolayer of CV-1 cells in duplicate petri dishes. SVV-infected cells were incubated at 37°C in CO2 for 4 to 5 days, and SVV plaques were fixed in methanol, stained with cresol violet, and counted. The titer of the antibody was determined on the basis of the dilution of serum that inhibited 80% or more of the plaques.
DNA extraction.
DNA was extracted from lymph node tissue samples as described previously (4, 14, 15).
Real-time qPCR.
DNA extracted from triplicate lymph node tissue samples was analyzed by real-time quantitative PCR (qPCR) using SVV open reading frame 61 (ORF61)-specific primers as described previously (5).
Rabbit polyclonal antibodies against SVV IE63.
Two synthetic peptides specific for the SVV IE63 protein were selected on the basis of their antigenic potential, based on analysis of hydrophobicity and accessibility parameters, and synthesized by Alpha Diagnostic International (San Antonio, TX). The two peptides, NH2-CIARASGRVTRDSRTLRR-COOH and NH2-CEHMAAKILTELRESVHNT-COOH, included amino acids 69 to 86 and 244 to 261, respectively. Rabbits were immunized three times with 5 mg of each peptide conjugated to a carrier protein. Antibody titers were determined by enzyme-linked immunosorbent assay.
Immunohistochemistry.
Sections (5 μm) of skin, lung, ganglionic, or lymph node tissue were analyzed for the presence of SVV glycoprotein H (gH) and gL, the IE63 protein, or SVV nucleocapsids by immunohistochemistry as described previously (13). Normal rabbit serum (1:2,000 to 15,000 dilution) or a mixture of polyclonal antipeptide antibodies against SVV gH and gL (1:2,000 dilution) or polyclonal antipeptide antibodies against the SVV IE63 protein (1:15,000) raised in rabbits were used for analysis (28). Each experiment was repeated at least 3 times.
Immunofluorescence analysis.
Fixed sections (5 μm) of lymph node tissues were deparaffinized in xylene for 5 min, followed by 3 rinses (5 min each) in 100% ethanol. Unless stated otherwise, all incubations were carried out at room temperature. Sections were dehydrated in graded ethanol and washed in phosphate-buffered saline (PBS) for 5 min. Antigen retrieval was performed by incubating the sections in 10 mM sodium citrate, pH 6.0, in a vegetable steamer (vapor phase) for 10 min and cooling at room temperature for 5 min. After washing once for 5 min in PBS containing 0.05% Tween 20 followed by blocking in 10% normal horse or goat serum for 30 min, the sections were rinsed once, washed in PBS for 5 min, and incubated at 4°C for 16 h with primary antibody (normal rabbit serum [1:2,000 to 1:5,000 dilution] with or without mouse IgG1 [catalog no. X0931; Dako] or IgG2a [catalog no. 554527; BD Pharmingen], SVV IE63-specific rabbit polyclonal antiserum [1:15,000 dilution], or a mixture of rabbit polyclonal antibody specific for SVV gH and gL [1:2,000 dilution] with or without mouse anti-human CD163 [catalog no. MCA1853; AbD Serotec]). Sections were washed 3 times for 5 min each time with PBS and further incubated for 2 h with secondary antibody (1:1,000 dilution of donkey Alexa Fluor 488-tagged anti-rabbit IgG [catalog no. A21206; Invitrogen] with or without a 1:1,000 dilution of goat Alexa Fluor 594-tagged anti-mouse IgG [catalog no. A11005; Invitrogen]). After washing 3 times for 3 min each time with PBS, the sections were fixed in 1% paraformaldehyde in PBS for 1 min, briefly washed twice with PBS, incubated with DAPI (4′,6-diamidino-2-phenylindole; 1:4,000 dilution of 1 mg/ml) for 2 min, washed 3 times with PBS, and mounted on glass coverslips using Prolong Gold antifade reagent (catalog no. P36930; Invitrogen). Sections were visualized using either a Nikon E800 fluorescence microscope or an Olympus FV1000 confocal microscope at the Advanced Light Microscopy Core Facility at the University of Colorado Anschutz Medical Campus.
RESULTS
Primary SVV infection, establishment of latency, and experimental reactivation in monkeys.
Seven Indian rhesus macaques inoculated with either wild-type SVV or SVV-EGFP developed viremia and varicella (with one exception) at 4 to 14 days postinoculation (dpi) (Fig. 1 and Table 2) and seroconverted at 28 dpi (Table 3). SVV-specific antibody titers remained high (1:320) in one monkey (monkey HI83) but were reduced (1:160) after dpi 142 in two monkeys (monkeys HB62 and HF39). In two monkeys, SVV antibody titers were reduced to 1:40 by dpi 142. SVV viremia peaked in all seven monkeys 4 to 7 dpi. Blood obtained from monkeys at 4 to 7 dpi had lower numbers of copies of SVV DNA than SVV-infected cells in culture, which required dilutions of up to 1012 to detect 1,000 copies of DNA by qPCR (data not shown). Five months later, after the establishment of latency, four monkeys (monkeys HB62, HI83, HF39, and HC44) were immunosuppressed; monkey HA95 was not immunosuppressed but had been transported with the others. Before immunosuppression, white blood cell (WBC) counts in all five monkeys were normal (7 × 103 to 10 × 103 cells per microliter). After immunosuppression, a gradual decline in WBC counts was observed and was reduced to 1 × 103 to 3 × 103 cells per microliter by day 176, confirming effective immunosuppression in all four treated monkeys. The untreated control monkey (monkey HA95) had 7.3 × 103 cells per microliter by day 176. At 7 to 12 weeks after immunosuppression, zoster lesions were observed in the left caudal abdomen in monkey HB62; the thorax, abdomen, and scrotum in monkey HI83; the thorax and the cranial and caudal abdomen in monkey HF39; the right arm and right and left arm in monkey HC44; and in the axilla (Fig. 2) and face in monkey HA95. Based on the findings of earlier studies, in which both immunosuppression and the stress of transportation and isolation produced SVV reactivation (4, 13), we believe that the stress of transportation and isolation resulted in SVV reactivation in monkey HA95. Neither viremia nor SVV antibody status changed dramatically in any monkey at the time of zoster (Tables 1 and 3).
TABLE 2.
Viremia after SVV inoculation and immunosuppression in rhesus macaques
| Monkey no. | SVV DNA copy no. on: |
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| dpia −18 | dpi 2 | dpi 4 | dpi 7 | dpi 9 | dpi 11 | dpi 14 | dpi 28 | dpi 42 | dpi 56 | dpi 151b | Day of zosterd | |
| HB62 | 0 | 0 | 6 | 145 | 43 | 11 | 13 | 2 | 1 | 3 | 0 | 0 |
| HI83 | 0 | 0 | 22 | 264 | 59 | 28 | 11 | 3 | 1 | 3 | 2 | 0 |
| HF39 | 0 | 0 | 0 | 37 | 21 | 5 | 17 | 6 | 2 | 2 | 0 | 0 |
| HC44 | 0 | 0 | 2 | 11 | 9 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| HA95c | 0 | 0 | 8 | 69 | 6 | 6 | 4 | 2 | 0 | 2 | 0 | 0 |
| II49 | 0 | NDe | 11 | 9 | 10 | ND | 4 | ND | ND | ND | ND | NAf |
| IK10 | 0 | ND | 5 | 2 | ND | 2 | 1 | ND | ND | ND | ND | NA |
dpi, day postinoculation.
One day after immunosuppression.
The monkey was not immunosuppressed.
Zoster rash was observed on dpi 197 (monkey HB62), 197 (monkey HI83), 217 (monkey HF39), 238 (monkey HC44), and 190 (monkey HA95).
ND, not done.
NA, not applicable.
TABLE 3.
Antibody response to SVV infection and immunosuppression in rhesus macaques
| Monkey no. | Titera at the following times: |
||||||
|---|---|---|---|---|---|---|---|
| Prebleed | dpib 28 | dpi 42 | dpi 56 | dpi 142 | dpi 155c | Necropsy | |
| HB62 | <1:10 | >1:320 | >1:320 | >1:320 | >1:320 | 1:160 | 1:160 |
| HI83 | <1:10 | >1:320 | >1:320 | >1:320 | >1:320 | >1:320 | >1:320 |
| HF39 | <1:10 | >1:320 | >1:320 | >1:320 | 1:160 | 1:160 | 1:160 |
| HC44 | <1:10 | >1:320 | 1:160 | 1:160 | 1:40 | 1:40 | 1:80 |
| HA95d | <1:10 | 1:160 | 1:320 | 1:320 | 1:40 | 1:80 | 1:40 |
| II49 | <1:10 | NDe | <1:320 | ND | ND | ND | 1:320 |
| IK10 | <1:10 | ND | 1:320 | ND | ND | ND | 1:320 |
| IE04 | NAf | NA | NA | NA | NA | NA | <1:10 |
Anti-SVV antibody titers are expressed as the serum dilution that neutralized at least 80% of SVV plaques compared to the amount neutralized in control cultures.
dpi, day postinoculation.
Five days after immunosuppression.
The monkey was not immunosuppressed.
ND, not done.
NA, not applicable.
FIG 2.
Zoster rash in the axilla of monkey HA95 at 6 months after primary infection.
Detection of SVV antigens in multiple tissues in monkeys after SVV reactivation.
The SVV IE63 protein was detected in the skin epithelium (not shown) and sweat glands of monkey HB62 (Fig. 3A) and in all other monkeys (data not shown). SVV gH and gL were detected in lung alveoli of monkey HB62 (Fig. 3C). SVV antigens were seen in both the nucleus and the cytoplasm of neurons and, less frequently, in nonneuronal cells in lumbar ganglia from monkey HC44 (Fig. 3E). SVV antigens were also detected in the ganglia of all other monkeys (data not shown). SVV antigens were detected in the medulla of the inguinal lymph nodes of monkey HC44 (Fig. 3G). In all five monkeys, the SVV IE63 protein was found in multiple lymph nodes, some of which were associated with areas of rash. While SVV antigens were found in all tissues, the amount detected in different monkeys was variable.
FIG 3.
Detection of SVV antigens in tissues from rhesus macaques after SVV reactivation. Paraformaldehyde-fixed, paraffin-embedded tissue sections were analyzed by immunohistochemistry. In monkey HB62, the SVV IE63 protein was detected in the sweat glands of skin at the site of zoster after immunostaining with rabbit polyclonal antibody directed against SVV IE63 peptides (A, arrow) and in lung alveoli immunostained with rabbit polyclonal antibody directed against SVV gH and gL (C, arrows). In monkey HC44, SVV antigen was seen in neurons and nonneuronal cells of lumbar ganglia (E, arrows) and in the medulla of inguinal lymph nodes (G, arrows) immunostained with rabbit polyclonal antibodies directed against SVV nucleocapsids. No staining was seen in the respective adjacent sections when preimmune rabbit serum was substituted for anti-SVV antibody (B, D, F, and H). Magnifications, ×600 (A and B) and ×200 (C to H).
Detection of SVV DNA in lymph nodes from monkeys after SVV reactivation.
On the basis of the findings of three independent PCRs, an average of 30 copies of SVV DNA was detected in 100 ng of DNA extracted from snap-frozen axillary lymph node from monkey HA95. No SVV-specific sequences were found in axillary lymph node DNA from SVV-seronegative monkey IE04.
Detection of SVV IE63 antigen in macrophages and dendritic cells in lymph nodes from monkeys after SVV reactivation.
The SVV IE63 protein was seen in the medulla of the inguinal lymph node from monkey HC44 after reactivation (Fig. 4B) but not in the inguinal lymph node from latently infected monkey II49 (Fig. 4F). Macrophages were found in the medulla of lymph nodes from both monkeys (Fig. 4C and G). The SVV IE63 protein colocalized with macrophages after reactivation (Fig. 4D) and was not seen in the lymph nodes from latently infected monkey II49 (Fig. 4H). The SVV IE63 protein was seen in the germinal center of the axillary lymph node from monkey HC44 after reactivation (Fig. 5B) and in dendritic cells in the germinal center (Fig. 5C). The SVV IE63 protein colocalized with dendritic cells in the axillary lymph node from monkey HC44 (Fig. 5D). Dendritic cells in the germinal center of the lymph node from latently infected monkey II49 (Fig. 5G) did not contain SVV IE63 (Fig. 5F).
FIG 4.
Detection of the SVV IE63 protein in macrophages in lymph nodes from rhesus macaques after virus reactivation but not during latency. Paraformaldehyde-fixed, paraffin-embedded sections of inguinal lymph node tissue from monkey HC44 after zoster (A to D) and from latently infected monkey II49 (E to H) were analyzed by immunofluorescence. In monkey HC44, the SVV IE63 protein was detected in macrophages (B and C) in the cortical sinus immunostained with rabbit polyclonal antibodies directed against SVV IE63 peptides and with mouse anti-human CD163 (macrophages) antibody. Donkey Alexa Fluor 488-tagged anti-rabbit IgG (H+L) antibody and goat Alexa Fluor 594-tagged anti-mouse IgG (H+L) antibody were used as secondary antibodies. In latently infected monkey II49, the SVV IE63 protein was not detected (F), although macrophages were found (G). Nuclei were stained with DAPI (A and E) and visualized at 358 nm. (D and H) Merged images. Magnifications, ×600.
FIG 5.
Detection of the SVV IE63 protein in dendritic cells in lymph nodes from rhesus macaques after virus reactivation but not during latency. Immunofluorescent staining of sections of paraformaldehyde-fixed, paraffin-embedded axillary lymph node tissue from monkey HC44 obtained 24 to 48 h after zoster (A to D) and inguinal lymph node tissue from latently infected monkey II49 (E to H). In monkey HC44, the SVV IE63 protein was detected in dendritic cells (B and C) in germinal centers immunostained with polyclonal antibodies directed against SVV IE63 peptides and mouse anti-human CD123 (dendritic cells). The fluorescence-tagged secondary antibodies used are described in the legend to Fig. 4. In latently infected monkey II49, the SVV IE63 protein was not detected (F), although dendritic cells were found (G). Nuclei were stained with DAPI (A and E). (D and H) Merged images. Magnifications, ×600.
Table 4 lists the results of analysis of multiple lymph nodes from all five monkeys after SVV reactivation, three lymph nodes from two monkeys latently infected with SVV (monkeys II49 and IK10), and three lymph nodes from one SVV-seronegative monkey (monkey IE04) using antibodies specific for the SVV IE63 protein and SVV gH and gL. SVV IE63 was detected in 12 of 18 (66%) lymph nodes, and SVV gH and gL were detected in all 17 (100%) lymph nodes from five monkeys after reactivation. SVV IE63, gH, and gL were not found in any lymph nodes from two latently infected monkeys or in lymph nodes from one SVV-seronegative monkey. Either the SVV IE63 protein or SVV gH and gL were detected in macrophages in 14 of 18 (77%) lymph nodes and in dendritic cells in 12 of 18 (67%) lymph nodes from five monkeys after reactivation. SVV antigens were absent in macrophages and dendritic cells in lymph nodes from two latently infected monkeys and one seronegative monkey.
TABLE 4.
Detection of SVV antigens in lymph nodes from rhesus macaques 24 h after appearance of zosterd
| Monkey no. | Lymph node type | Detection of the following antigen: |
|||
|---|---|---|---|---|---|
| SVV IE63 | SVV gH and gL | CD163 (Mϕ) | CD123 (DC) | ||
| HB62 | Axillary | + | + | + | + |
| Bronchial | − | + | − | − | |
| Inguinal | + | + | + | + | |
| Mandibular | − | + | + | − | |
| HI83 | Axillary | + | + | + | + |
| Bronchial | + | + | + | + | |
| Inguinal | + | + | + | + | |
| HF39 | Axillary | + | + | + | + |
| Bronchial | − | + | − | − | |
| Iliac | + | + | + | + | |
| Inguinal | − | + | + | + | |
| HC44 | Axillary | + | + | + | + |
| Bronchial | − | ND | − | − | |
| Inguinal | + | + | + | + | |
| HA95a | Axillary | + | + | + | + |
| Bronchial | − | + | − | − | |
| Inguinal | + | + | + | + | |
| Mandibular | + | + | + | − | |
| II49b | Axillary | − | − | − | − |
| Inguinal | − | − | − | − | |
| IK10b | Axillary | − | − | − | − |
| IE04c | Axillary | − | − | − | − |
| Bronchial | − | ND | − | − | |
| Inguinal | − | − | − | − | |
Not immunosuppressed.
The monkey was latently infected.
The monkey was SVV seronegative.
ND, not done; Mϕ, macrophages; DC, dendritic cells.
Subcellular localization of SVV antigens in macrophages and dendritic cells in lymph nodes from monkeys after SVV reactivation.
Confocal microscopic analysis of lymph node tissues after reactivation revealed the SVV IE63 protein in the cytoplasm of macrophages (Fig. 6D) and dendritic cells (Fig. 7D).
FIG 6.
Detection of the SVV IE63 protein in the cytoplasm of macrophages in lymph nodes from a rhesus macaque after virus reactivation. Confocal microscopy revealed the SVV IE63 protein in the cytoplasm of macrophages (D, arrow) immunostained with the antibodies described in the legend to Fig. 4 in sections of inguinal lymph node tissue from monkey HC44 obtained 24 to 48 h after zoster. Nuclei were stained with DAPI (A). (D) Merged image. Magnifications, ×630.
FIG 7.
Detection of the SVV IE63 protein in the cytoplasm of dendritic cells in lymph nodes from a rhesus macaque after virus reactivation. Confocal microscopy revealed the SVV IE63 protein in the cytoplasm of dendritic cells in sections of axillary lymph node tissue from monkey HC44 (D, arrow) obtained 24 to 48 h after zoster and immunostained with the same antibodies described in the legend to Fig. 5. Nuclei were stained with DAPI (A). (D) Merged image. Magnifications, ×600.
Detection of SVV IE63 protein in CD3+ T cells in lymph nodes from monkeys after SVV reactivation.
The SVV IE63 protein was also seen in CD3+ T cells located in the lymphatic nodule of the inguinal lymph node from monkey HC44 (Fig. 8D). Fewer CD3+ T cells than macrophages and dendritic cells contained the SVV ORF63 antigen. SVV antigens did not colocalize with B cells in the inguinal lymph node from monkey HC44 (data not shown). Overall, after experimental reactivation, SVV antigens were found in the skin, lungs, and ganglia and in the cytoplasm of macrophages, dendritic cells, and T cells.
FIG 8.
Detection of the SVV IE63 protein in CD3+ T cells in lymph nodes from rhesus macaques after virus reactivation but not during latency. Sections of inguinal lymph node tissue from monkey HC44 obtained 24 to 48 h after zoster (A to D) and from latently infected monkey II49 (E to H) were analyzed by immunofluorescence. In monkey HC44, the SVV IE63 protein was detected in CD3+ T cells after immunostaining with rabbit polyclonal antibodies directed against SVV IE63 peptides (B) and mouse anti-human CD3 (T cells) antibody (C). The fluorescence-tagged secondary antibodies used are described in the legend to Fig. 4. In latently infected monkey II49, the SVV IE63 protein was not detected (F), although CD3+ T cells were found (G). Nuclei were stained with DAPI (A and E) and visualized at 358 nm. (D and H) Merged images. Magnifications, ×600.
DISCUSSION
The exact mechanism by which varicella reactivates from ganglia and is transported to skin is unknown. SVV infection in nonhuman primates provides a model system with which to identify cells involved in the dissemination of varicella to multiple organs during primary infection as well as after reactivation. During experimental primary SVV infection in AGM, alveolar macrophages and dendritic cells become infected at 5 dpi and transfer virus to T cells in lymph nodes (12). In the study described here, we extended studies of SVV reactivation in rhesus macaques and demonstrated the presence of SVV in the sweat glands of skin and in the macrophages and dendritic cells of lymph nodes. Compared to the low levels seen in the ganglia and lungs, lymph nodes contained an abundance of SVV antigen (Fig. 3), and this finding was validated by the detection of SVV DNA. Importantly, in VZV-infected neurons in vitro, VZV DNA is not abundant, despite extensive VZV transcription and translation (16). While SVV antigens were seen in the skin, lung, ganglionic, and lymph node tissues of all five monkeys, a stronger signal was found in tissues from monkeys HC44 and HB62 than in those from the other monkeys.
Detection of SVV antigens in macrophages that express both CD163 and CD68 suggests phagocytosis, since CD163 is expressed on activated macrophages, while CD68 is a marker for cells of the macrophage lineage (17). Dendritic cells and macrophages take up antigens in the periphery and display major histocompatibility complex-peptide complexes to T cells in lymphoid organs (18). Thus, detection of SVV antigens in both lymph node macrophages and lymph node dendritic cells after reactivation is consistent with the notion that these cells present antigens to T cells in lymph nodes.
Since T cell numbers increase at the time of zoster in immunosuppressed monkeys (19) and lymph nodes receive afferent neural input from the ganglia (20, 21), analysis of ganglionic and lymph node tissues for virus DNA and antigens from monkeys within hours after SVV-specific T cell levels in blood are increased will help to determine whether reactivated SVV is transported transaxonally from the ganglia to the lymph nodes. Indeed, detection of SVV antigens in sweat glands (Fig. 3A) in the absence of viremia most likely reflects the transaxonal transport of reactivated SVV from the ganglia to the skin.
Although focal necrosis, multinucleated epithelial cells, and viral infection of the sweat glands are seen in the affected skin of humans during zoster (22, 23), we did not find these pathological features in the skin of monkeys with zoster. The absence of SVV viremia during zoster in monkeys parallels the findings in humans with varicella and zoster (24–26).
Finally, since VZV infection of dendritic cells in culture is productive (27), it will be interesting to examine possible SVV infection of lymph node macrophages and dendritic cells from monkeys after zoster.
ACKNOWLEDGMENTS
We thank Wayne Gray for providing the SVV gH and gL antibodies, the Tulane National Primate Research Center Veterinary Medicine staff for excellent animal care, Georges Verjans and Werner Ouwendijk for useful discussions, Marina Hoffman for editorial assistance, and Cathy Allen for manuscript preparation.
This work was supported in part by Public Health Service grants AG032958 (to D.G., R.M., and V.T.-D.) and AG006127 (D.G.) from the National Institutes of Health. This work was also supported in part with federal funds from the National Center for Research Resources and the Office of Research Infrastructure Programs (ORIP) of the National Institutes of Health through grant number P51 RR00164 to the Tulane National Primate Research Center (to V.T.-D.). Imaging experiments were performed in the University of Colorado Anschutz Medical Campus Advanced Light Microscopy Core Facility, supported in part by NIH/NCRR Colorado CTSI grant number UL1 RR025780.
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