Abstract
Simian varicella virus (SVV) infection of rhesus macaques (RMs) recapitulates the hallmarks of varicella-zoster virus (VZV) infection of humans, including the establishment of latency within the sensory ganglia. Various factors, including age and immune fitness, influence the outcome of primary VZV infection, as well as reactivation resulting in herpes zoster (HZ). To increase our understanding of the role of lymphocyte subsets in the establishment of viral latency, we analyzed the latent SVV transcriptome in juvenile RMs depleted of CD4 T, CD8 T, or CD20 B lymphocytes during acute infection. We have previously shown that SVV latency in sensory ganglia of nondepleted juvenile RMs is associated with a limited transcriptional profile. In contrast, CD4 depletion during primary infection resulted in the failure to establish a characteristic latent viral transcription profile in sensory ganglia, where we detected 68 out of 69 SVV-encoded open reading frames (ORFs). CD-depleted RMs displayed a latent transcriptional profile that included additional viral transcripts within the core region of the genome not detected in control RMs. The latent transcriptome of CD20-depleted RMs was comparable to the latent transcription in the sensory ganglia of control RMs. Lastly, we investigated the impact of age on the establishment of SVV latency. SVV gene expression was more active in ganglia from two aged RMs than in ganglia from juvenile RMs, with 25 of 69 SVV transcripts detected. Therefore, immune fitness at the time of infection modulates the establishment and/or maintenance of SVV latency.
INTRODUCTION
Simian varicella virus (SVV) and varicella-zoster virus (VZV) are neurotropic alphaherpesviruses that establish latency within the sensory ganglia during primary infection. Viral latency is generally characterized by a quiescent state with limited gene expression; for example, during herpes simplex virus (HSV) latency, only the latency-associated transcripts (LATs) are abundantly expressed (1). However, VZV latency can be associated with the transcription of multiple genes, including open reading frames (ORFs) 4, 11, 18, 21, 29, 40, 41, 43, 57, 62, 63, 66, and 68 (2–9). We recently examined the SVV latent transcriptome from juvenile rhesus macaques (RMs) and found that SVV latency is characterized by a restricted gene expression profile (10). SVV ORF 61 was the most prevalent transcript detected during latent infection, present in at least one sensory ganglion in all RMs tested and in 10 of 16 sensory ganglia total. We also detected SVV ORFs A, B, 4, 10, 55, 63, 64, 65, 66, 67, and 68, although less frequently, in 1 to 4 of 16 latently infected sensory ganglia from juvenile RMs.
Several clinical observations highlight the importance of cell-mediated immune responses in controlling VZV infection and reactivation. Specifically, a lack of immunoglobulin production due to agammaglobulinemia does not complicate the outcome of varicella in children (11, 12). In contrast, T cell deficiencies, including congenital deficiencies or those induced by HIV infection or immune suppression, lead to severe and disseminated varicella (13–17). Similarly, a higher incidence of herpes zoster (HZ) in aged patients is associated with diminished T cell proliferation to VZV antigens in vitro, while antibody titers remain stable (18). Moreover, HIV patients are more susceptible to HZ when their absolute numbers of CD4 T cells decline to less than 500 cells per microliter (19–21). Finally, the frequency of VZV reactivation is related to the intensity of immune suppression, with higher incidence in patients receiving combined therapy than in patients receiving chemotherapy or radiotherapy alone (22).
We have recently shown that the resolution of acute SVV infection is also dependent on cellular immunity. Specifically, loss of CD4 T cells during acute SVV infection in juvenile RMs resulted in higher peak viral loads, prolonged viremia, and disseminated varicella compared to these parameters in controls (23). CD8-depleted RMs had slightly higher viral loads and more prolonged varicella rash than controls. Lastly, CD20 depletion did not alter the severity of varicella or the kinetics and magnitude of the T cell response. Our data indicate that CD4 T cell immunity is critical in controlling acute SVV infection in RMs and are in agreement with clinical observations during acute VZV infection.
To improve HZ-associated morbidity, it is critical to understand the virological and immunological parameters that control latency and reactivation. In the present study, we extend our previous reports (10, 23) and investigate the impact of T cell and B cell loss and advanced age during acute infection on latent SVV transcriptional profiles. Our data show that CD4 T cells are important, whereas CD8 T cells and B cells have a minimal impact on the establishment of SVV latency. Similar to the loss of CD4 T cells, acute infection of aged RMs also has a significant effect on SVV gene expression in sensory ganglia during latency.
MATERIALS AND METHODS
Cells and virus.
Simian varicella virus (SVV; Cercopithecine herpesvirus 9) was propagated in telomerized rhesus fibroblasts (tRFs) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin–streptomycin–l-glutamine (PSG). SVV-infected tRFs were harvested at the height of cytopathic effect by scraping, frozen in FetalPlex (FBS alternative) (Gemini Bio-products, Sacramento CA) supplemented with 10% dimethyl sulfoxide (DMSO), and assayed by plaque assay on primary rhesus fibroblasts maintained in DMEM supplemented with 10% FBS and PSG.
Animals and sample collection.
All rhesus macaques were housed at the Oregon National Primate Research Center and were handled in accordance with good animal practices as defined by the Office of Laboratory Animal Welfare. Animal work was approved by the Oregon National Primate Research Center Institutional Animal Care and Use Committee. Immune cell-depleted juvenile (3 to 4 years old) rhesus macaques (RM; Macaca mulatta) were previously described (23). Briefly, RMs 24933, 24952, 25111, and 25152 were depleted of CD4 T cells by administering the humanized monoclonal antibody OKT4-HulgG (NIH Nonhuman Primate Reagent Resource) at −3, 0, and 7 days postinfection (d.p.i.) at a dose of 50 mg/kg of body weight. RMs 25343, 25371 26108, and 26842 were depleted of CD8 T cells via the administration of mouse-human chimeric monoclonal antibody cM-T807 (NIH Nonhuman Primate Reagent Resource) at −3 d.p.i. at a dose of 10 mg/kg and at 0, 3, and 7 d.p.i. at a dose of 5 mg/kg. RMs 25833, 25892, 25905, and 25920 were depleted of CD20 B cells using the mouse-human chimeric antibody Rituxan (Genentech, San Francisco CA) given at −7, 0, and 7 d.p.i. at 20 mg/kg. Four animals (20266, 24943, 24953, and 25043) were left untreated as nondepleted controls (10, 23). Aged RMs 15461 and 15449 were 19 years old. RMs were SVV seronegative prior to infection, as tested by enzyme-linked immunosorbent assay (ELISA) (23, 24). All animals were infected intrabronchially with 4 × 105 PFU SVV. Animals were euthanized 63 to 100 days postinfection. Intact sensory ganglia, including trigeminal ganglia (TG) and cervical, thoracic, and lumbar-sacral dorsal root ganglia (DRG-C, DRG-T, and DRG-L/S, respectively), were divided, flash frozen, and stored at −80°C until analysis. Whole ganglia were allocated proportionally, one part for viral load analysis and one part for transcriptional analysis, except for the TG, which because of size was only used for transcriptional analysis.
DNA extraction and qPCR.
DNA was extracted from heparinized whole blood (WB), bronchoalveolar lavage (BAL) fluid, and portions of frozen ganglia using an ArchivePure DNA cell/tissue kit (5 PRIME, Gaithersburg, MD) according to the manufacturer's protocol. SVV DNA viral loads in WB, BAL fluid, and sensory ganglia were measured by quantitative real-time PCR (qPCR) using Maxima probe/ROX qPCR master mix (2×) (Fermentas, Glen Burnie MD) and primers/TaqMan probe specific for SVV ORF 21. Following an initial 10-min, 95°C step, 40 cycles of 15 s at 95°C and 1 min at 60°C were completed using a StepOnePlus (Life Technologies, Grand Island NY). SVV bacterial artificial chromosome (BAC) DNA was used as the quantification standard (25).
RNA isolation and amplification.
Sensory ganglion total RNA was isolated from latently infected rhesus macaques using the Isol-RNA lysis reagent (5 PRIME) method, and samples were DNase treated using the Turbo DNA-free kit (Ambion, Austin TX). RNA amplification was performed using one of two methods, as follows. (i) In the 3-round amplification protocol, first-strand cDNA was synthesized from 1 μg of total RNA using 0.5 μM T7 oligo(dT)-T7 primer and 250 U SuperScript III reverse transcriptase (Life Technologies). Double-stranded cDNA was generated by the addition of second-strand buffer (Life Technologies) according to the manufacturer's instructions. Double-stranded cDNA was purified using phenol-chloroform-isoamyl alcohol extraction and concentrated in Amicon ultra centrifugal filters (Millipore, Billerica MA) before being amplified using the T7 MEGAscript kit (Ambion) according to the manufacturer's method. The amplified RNA (aRNA) was purified using the RNeasy minikit (Qiagen, Valencia CA), and 5 μg of aRNA was reverse transcribed with 350 U SuperScript III reverse transcriptase in the presence of 9 μg of random primers (Life Technologies). Double-stranded cDNA and second-round aRNA were generated as described above, repeating for a total of three rounds of RNA amplification. (ii) In the global PCR amplification protocol, RNA was amplified using global PCR and T7 RNA polymerase-based amplification adapted from references 26, 27 and 28. Briefly, first-strand cDNA was synthesized from 1 μg of total RNA using T7 oligo(dT)-T7 primer and SuperScript III reverse transcriptase (Life Technologies). Second-strand synthesis was performed using Taq DNA polymerase (New England BioLabs, Ipswich, MA) and a degenerate oligonucleotide (DOP) primer (500 ng/μl CCGACTCGAGNNNNNNATGTGG) using a PCR program consisting of 94°C for 3 min and 30°C for 2 min and then increasing the heat from 30°C to 72°C with a rate increase of 0.2°C/s and holding at 72°C for 4 min. Next, global PCR was performed with the addition of T7 oligo(dT) and DOP primers, followed by 25 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 4 min, followed by a hold at 72°C for 6 min. The cDNA was isolated using phenol chloroform extraction and concentrated with the DNA Clean & Concentrator-5 kit (Zymo Research, Irvine CA). The cDNA was amplified using the T7 MEGAscript kit (Ambion), and the aRNA was purified using the RNeasy minikit (Qiagen). Lastly, using either amplification protocol, 10 μg of aRNA was reverse transcribed using the high-capacity cDNA reverse transcription kit (Life Technologies). The three-round amplification protocol was performed for nondepleted juveniles (10), CD4-depleted RMs 24952 and 24993, and aged RMs 15449 and 15461. The global PCR amplification protocol was performed for CD4-depleted RMs 25111 and 25152 and all CD8- and CD20-depleted RMs. Both methods started with 1 μg of total RNA, and amplification was not statistically different between these two methods as determined by Student's t test (P = 0.32). The average amplification for three-round amplification was 85.6 ± 25.1 μg (mean ± standard deviation [SD]), and the range of amplification was 33.2 to 134.1 μg. The average amplification for global PCR amplification was 90.7 ± 13.7 μg, and the range of amplification was 70.4 to 121.4 μg.
RT-qPCR.
cDNA was analyzed by quantitative real-time reverse transcriptase PCR (RT-qPCR) using primer and probe sets specific for each ORF in the SVV genome. A list of the primer and probe sequences designed using Primer Express software (Life Technologies) for SVV (10) and for the reference gene, encoding M. mulatta glutathione synthetase (29), was previously described. RT-qPCR was performed using specific TaqMan probes and 2× Gene Expression master mix (Life Technologies) or Maxima probe/ROX qPCR master mix (2×) (Fermentas). Following an initial 10-min, 95°C step, 40 cycles of 15 s at 95°C and 1 min at 60°C were completed using a StepOnePlus RT-qPCR machine (Life Technologies). Plasmids containing each target amplicon, synthesized amplicons, or SVV BAC DNA were serially diluted, covering five log10 concentrations, and used to construct standard curves for relative quantification. A positive signal in our analysis required that any given SVV gene express at least 100 copies in amplified RNA from 1 μg of total RNA from ganglion samples using either amplification protocol. Copy numbers are reported as the average of triplicate RT-qPCR analyses for each sample and are within 25% standard deviation.
Intracellular cytokine staining.
BAL fluid cells were stimulated with SVV lysate (1 μg) for 1 h, followed by the addition of brefeldin A (Sigma, St. Louis, MO) for 14 h. After stimulation, cells were surface stained with antibodies against CD4 (eBioscience, San Diego, CA), CD28, and CD95 (BioLegend, San Diego, CA) to delineate central memory (CD28+ CD95+) and effector memory (CD28− CD95+) T cell subsets. Samples were fixed, permeabilized (BioLegend), and dual stained using antibodies against gamma interferon (IFN-γ) (eBioscience) and tumor necrosis factor alpha (TNF-α) (eBioscience). Samples were analyzed using the LSRII instrument (Becton, Dickinson and Company, San Jose CA) and FlowJo software (TreeStar, Ashland OR).
ELISA.
ELISA plates were coated with SVV lysate overnight at 4°C, blocked with 5% milk in wash buffer (0.05% Tween in PBS) for 1 h at room temperature, washed three times with wash buffer, and incubated with heat-inactivated (55°C for 30 min) plasma samples in 3-fold dilutions in duplicate for 1 h. After washing three times with wash buffer, horseradish peroxidase (HRP)-conjugated anti-rhesus IgG (Nordic Immunology, Netherlands) was added for 1 h, followed by the addition of chromogen o-phenylenediamine · 2HCl (OPD) (Sigma, St. Louis, MO) substrate for 20 min to allow the detection and quantification of bound antibody molecules. The reaction was stopped with the addition of 1 M HCl. The optical density was measured at 490 nm using an ELISA plate reader (SpectraMax 190; Molecular Devices, Sunnyvale, CA). Endpoint IgG titers were calculated using log-log transformation of the linear portion of the curve, with 0.1 optical density (OD) units as the cutoff. Titers were standardized using a positive-control sample included with each assay.
Statistical analysis.
Statistical analysis and graphing were conducted with GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA). The data in Figure 6 were analyzed by repeated measures of analysis of variance (ANOVA), with the Bonferroni posttest to explore differences between groups (juvenile RMs and aged RMs), at each time point. Logistic regression was used to determine statistical significance for the differences in incidence of ORF expression between depleted and aged cohorts compared to controls.
Fig 6.
Aged RMs experience transient viremia and delayed SVV-specific T cell response and IgG titer. (A and B) SVV DNA viral loads in BAL fluid (A) and whole blood (B) were measured by quantitative PCR using primers and probe specific for SVV ORF 21 from juvenile RMs and aged RMs 15449 and 15461. Dashed line indicates limit of detection. (C and D) Frequencies of SVV-specific CD4 central memory (CM) (C) and CD4 effector memory (EM) (D) T cells in BAL fluid producing IFN-γ, TNF-α, and IFN-γ/TNF-α was measured by intracellular cytokine staining following stimulation with SVV lysate. Data for juvenile RMs and aged RMs 15449 and 15461 are shown. (E) SVV-specific IgG antibody endpoint titers were measured by standard ELISA. Values are the average ± SEM. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.
RESULTS
CD4, CD8, and CD20 cell counts and viral loads at necropsy.
To characterize the impact of lymphocyte loss during acute simian varicella virus (SVV) infection on latent SVV gene expression profiles, we analyzed sensory ganglia collected from 12 rhesus macaques (RMs, aged 3 to 5 years, n = 4 per group) that were depleted of CD4 T, CD8 T, or CD20 B cells during acute infection (23). Circulating CD8 T and CD20 B cells were undetectable between 0 and 14 d.p.i., and circulating CD4 T cells were significantly reduced between 7 and 17 d.p.i. (23). All animals were infected intrabronchially with 4 × 105 PFU wild-type (WT) SVV. Disease severity, viral loads, and the host immune response during acute SVV infection of the RMs were previously published (23, 24). Briefly, CD4-depleted animals experienced the most severe disease and highest viral loads, whereas CD8-depleted animals had slightly worse clinical symptoms, and CD20-depleted animals exhibited viral loads and clinical symptoms that were indistinguishable from those in nondepleted controls (23). Animals were euthanized at 63 to 100 d.p.i., and sensory ganglia were collected (Table 1). At the time of necropsy, all RMs displayed undetectable SVV genome viral loads within whole blood and bronchoalveolar lavage (BAL) fluid cells and did not have a rash (23, 24). The numbers of T and B cells per microliter of blood at the time of necropsy are shown in Table 1. Fold change values were calculated for each lymphocyte subset from whole blood counts prior to depletion and infection compared to the counts at necropsy (Table 1). Within the CD4-depleted cohort, 24952 and 24993 at the time of necropsy sustained more than 4-fold decreases in CD4+ cells, while 25111 maintained a moderate decrease (2.7-fold), and CD4+ cells in 25152 had returned to baseline numbers. Within the CD8-depleted cohort, at necropsy, 26842 maintained a 3.5-fold decrease in CD8+ cells. Animals 25343 and 25371 retained moderate decreases (2.2- and 2.6-fold, respectively), and 26108 had returned to baseline levels of CD8+ cells. All RMs within the CD20-depleted cohort had CD20+ cell numbers at or near baseline levels (range, 0.8- to 1.5-fold change) at the time of necropsy.
Table 1.
Absolute numbers of cell populations in whole blood
| Group | Animal | Absolute no. of cells/μl WBa at: |
Fold change in no. of cellsb |
Time of necropsy (d.p.i.) | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Baseline |
Necropsy |
||||||||||
| CD4 | CD8 | CD20 | CD4 | CD8 | CD20 | CD4 | CD8 | CD20 | |||
| Control | 24943 | 853 | 430 | 239 | 1,564 | 952 | 802 | 0.5 | 0.5 | 0.3 | 77 |
| 24953 | 1,871 | 535 | 1,362 | 1,427 | 513 | 588 | 1.3 | 1 | 2.3 | 63 | |
| 25043 | 2,506 | 990 | 5,411 | 2,654 | 1,849 | 3,071 | 0.9 | 0.5 | 1.8 | 63 | |
| 25339 | 526 | 119 | 256 | 605 | 270 | 707 | 0.9 | 0.4 | 0.4 | 77 | |
| CD4 depleted | 24952 | 917 | 228 | 941 | 176 | 404 | 555 | 5.2 | 0.6 | 1.7 | 63 |
| 24993 | 835 | 341 | 1,222 | 180 | 1,117 | 315 | 4.6 | 0.3 | 3.9 | 63 | |
| 25111 | 587 | 146 | 168 | 214 | 1,234 | 1,415 | 2.7 | 0.1 | 0.1 | 86 | |
| 25152 | 849 | 285 | 775 | 847 | 1,431 | 2,442 | 1 | 0.2 | 0.3 | 85 | |
| CD8 depleted | 25343 | 589 | 165 | 44 | 789 | 76 | 473 | 0.7 | 2.2 | 0.1 | 100 |
| 25371 | 879 | 270 | 229 | 1,169 | 104 | 696 | 0.8 | 2.6 | 0.3 | 88 | |
| 26108 | 451 | 226 | 175 | 928 | 185 | 1,076 | 0.5 | 1.2 | 0.2 | 87 | |
| 26842 | 903 | 190 | 225 | 632 | 54 | 233 | 1.4 | 3.5 | 1 | 100 | |
| CD20 depleted | 25833 | 970 | 238 | 1,452 | 831 | 409 | 957 | 1.2 | 0.6 | 1.5 | 93 |
| 25892 | 739 | 332 | 99 | 281 | 162 | 88 | 2.6 | 2 | 1.1 | 91 | |
| 25905 | 973 | 248 | 167 | 424 | 114 | 203 | 2.3 | 2.2 | 0.8 | 93 | |
| 25920 | 949 | 385 | 1,067 | 948 | 730 | 745 | 1 | 0.5 | 1.4 | 91 | |
| Aged | 15461 | 1,013 | 1,506 | 346 | 2,212 | 845 | 634 | 0.5 | 1.8 | 0.5 | 64 |
| 15449 | 1,789 | 1,161 | 328 | 1,506 | 1,130 | 763 | 1.2 | 1 | 0.4 | 64 | |
Calculated by converting frequency of subset using complete blood counts obtained prior to treatment or infection (baseline) and at necropsy.
Fold change was calculated as count at baseline divided by count at necropsy.
We measured SVV viral load in sensory ganglia by quantitative real-time PCR using primers and probes specific for ORF 21 (Table 2). Ganglia were pooled at necropsy and portioned for multiple analyses, and thus, we were unable to measure viral loads from the same portions used for transcriptional profiles. Since SVV does not infect all sensory ganglia equally, the viral loads in Table 2 represent a snapshot of SVV DNA in that portion at the time of necropsy. Viral DNA was detectable in a majority of the ganglia tested. Specifically, SVV DNA was detected in 9 of 12 ganglia obtained from four CD4-depleted RMs, with a range of 1.30 × 101 to 2.09 × 104 copies per μg of DNA (average ± SD, 3.41 × 103 ± 6.46 × 103). We detected SVV DNA in 10 of 12 ganglia from the four CD8-depleted RMs, with a range of 3.30 × 101 to 2.41 × 103 copies per μg of DNA (average ± SD, 4.58 × 102 ± 7.15 × 102). Within the CD20-depleted cohort (n = 4), SVV DNA was detected in 9 of 12 ganglia, and the range of viral genome copies was 3.60 × 101 to 1.53 × 103 per μg of DNA (average ± SD, 5.64 × 102 ± 5.06 × 102).
Table 2.
SVV DNA viral load
| Group, animal | Sample typea | Copy no.b |
|---|---|---|
| CD4 depleted | ||
| 24952 | DRG-C | 5.02 × 101 |
| DRG-T | ND | |
| DRG-L/S | 1.09 × 103 | |
| 24993 | DRG-C | 1.06 × 103 |
| DRG-T | 5.73 × 102 | |
| DRG-L/S | 1.31 × 101 | |
| 25111 | DRG-C | ND |
| DRG-T | 6.24 × 103 | |
| DRG-L/S | 2.09 × 104 | |
| 25152 | DRG-C | ND |
| DRG-T | 6.88 × 102 | |
| DRG-L/S | 1.74 × 101 | |
| CD8 depleted | ||
| 25343 | DRG-C | 1.01 × 102 |
| DRG-T | 1.57 × 102 | |
| DRG-L/S | ND | |
| 25371 | DRG-C | 2.14 × 102 |
| DRG-T | 2.66 × 102 | |
| DRG-L/S | 7.04 × 101 | |
| 26108 | DRG-C | 1.10 × 103 |
| DRG-T | 2.41 × 103 | |
| DRG-L/S | ND | |
| 26842 | DRG-C | 1.15 × 102 |
| DRG-T | 3.28 × 101 | |
| DRG-L/S | 1.10 × 102 | |
| CD20 depleted | ||
| 25833 | DRG-C | 1.53 × 103 |
| DRG-T | 1.19 × 103 | |
| DRG-L/S | 8.69 × 101 | |
| 25892 | DRG-C | 5.83 × 102 |
| DRG-T | 3.65 × 101 | |
| DRG-L/S | 1.87 × 102 | |
| 25905 | DRG-C | ND |
| DRG-T | 5.46 × 102 | |
| DRG-L/S | 6.62 × 101 | |
| 25920 | DRG-C | ND |
| DRG-T | ND | |
| DRG-L/S | 8.49 × 102 | |
| Aged | ||
| 15461 | DRG-C | 2.27 × 101 |
| DRG-T | ND | |
| DRG-L/S | ND | |
| 15449 | DRG-C | 3.98 × 101 |
| DRG-T | ND | |
| DRG-L/S | ND |
DRG-C, cervical dorsal root ganglia; DRG-T, thoracic dorsal root ganglia; DRG-L/S, lumbar/sacral dorsal root ganglia.
Average copy number per μg of DNA. ND, not detected.
Efficiency of RNA amplification and RT-qPCR.
To measure the latent SVV transcriptional profile, total RNA was isolated from trigeminal ganglia (TG), cervical dorsal root ganglia (DRG-C), thoracic dorsal root ganglia (DRG-T), and lumbar/sacral dorsal root ganglia (DRG-L/S). For each ganglion, 1 μg of total RNA was amplified, and both total RNA and amplified RNA were analyzed by quantitative real-time reverse transcriptase PCR (RT-qPCR) using a primer and probe set specific for the reference gene, encoding glutathione synthetase (GSS) of M. mulatta. All 63 ganglia contained amplifiable RNA using either the three-round or the global amplification protocol (detailed in Materials and Methods; data not shown). The average quantification cycle (Cq) value ± SD for total RNA using the three-round amplification protocol was 23.0 ± 1.9, and for amplified RNA, it was 24.1 ± 1.9. The average Cq value ± SD for total RNA using the global PCR amplification protocol was 27.7 ± 3.3, and for amplified RNA, it was 28.5 ± 3.6.
Quantification of SVV transcripts was determined by linear regression of three technical replicates of each standard template dilution covering five log10 concentrations (10 to 1 × 106 copies) for each specific ORF primer/probe set (n = 69). All SVV ORFs were amplified with an efficiency of 90 to 110% except for ORFs 18, 19, 36, 37, 50, 62, and 64, which had a range of efficiencies from 75% to 123% (data not shown). Figure 1 is a compilation of all individual qPCR data points for each standard quantity using all SVV primer/probe sets (average ± SD). The overall standard curve PCR efficiency was 97.5 ± 7.5%. The slope of the linear regression line was −3.4 ± 0.20, with an R2 value of 0.99 (Fig. 1).
Fig 1.
Determination of efficiency of SVV DNA standard quantification using linear regression covering five log10 concentrations (10 to 1 × 106 copies). Specific primer pairs and TaqMan probes for all 69 SVV ORFs were analyzed in triplicate. Average Cq value ± standard deviation (SD) was obtained for each SVV DNA copy number. Data represent the combination of all individual qPCR data points for each standard quantity.
Impact of T and B cell depletion during acute infection on SVV transcriptional profiles in sensory ganglia.
Our previous transcriptional analysis of latently infected ganglia from nondepleted juvenile control RMs showed a restricted latent gene expression profile, detecting SVV ORFs from the left and right termini of the genome (10). An example of a latent gene expression profile from our previous study characterizing transcription in juvenile RMs is shown in Figure 2. The viral loads, transcriptional profiles, and gene expression values in latently infected sensory ganglia from control RMs were previously published (10). In summary, SVV ORF 61 was the most prevalent transcript detected during latent infection and was detected in at least one ganglion from all RMs analyzed. Transcripts from SVV ORFs A, B, 4, 10, 55, 63, 64, 65, 66, 67, and 68 were also detected, but the frequency of detection was substantially reduced compared to the frequency of ORF 61 in latently infected sensory ganglia (Table 3).
Fig 2.
SVV transcriptome in sensory ganglia of latently infected nondepleted juvenile RM 25043. Total RNA was isolated from trigeminal ganglia (TG), cervical dorsal root ganglia (DRG-C), thoracic dorsal root ganglia (DRG-T), and lumbar/sacral dorsal root ganglia (DRG-L/S). RNA was amplified and analyzed by RT-qPCR using primer and probe sets specific for each viral ORF. Data shown are the average copy numbers for triplicate reactions per sample from 1 μg of total RNA (10).
Table 3.
Prevalence of SVV gene expression during latency
| ORF | No. of positive samples from indicated group |
||||
|---|---|---|---|---|---|
| CD4 depleted (n = 12 samples; P < 0.0001a) | CD8 depleted (n = 14 samples; P = 0.0733) | CD20 depleted (n = 13 samples; P = 0.6021 | Aged (n = 8 samples; P < 0.0001 | Control (n = 16 samples)b | |
| A | 8 | 4 | 4 | 1 | 3 |
| B | 8 | 2 | 2 | 2 | 4 |
| 1 | 5 | 1 | 1 | 2 | |
| 2 | |||||
| 3 | 6 | 1 | 1 | 2 | |
| 4 | 7 | 1 | 2 | 1 | |
| 5 | 4 | 2 | |||
| 6 | 3 | ||||
| 7 | 5 | 3 | 3 | 1 | |
| 8 | 1 | 1 | |||
| 9 | 3 | ||||
| 9A | 3 | 3 | 2 | ||
| 10 | 2 | 1 | |||
| 11 | 5 | 1 | 1 | ||
| 12 | 1 | 1 | |||
| 13 | 7 | 1 | |||
| 14 | 4 | 5 | |||
| 15 | 7 | 2 | |||
| 16 | 4 | ||||
| 17 | 5 | ||||
| 18 | 6 | 2 | |||
| 19 | 2 | 1 | |||
| 20 | 4 | 1 | |||
| 21 | 2 | ||||
| 22 | 2 | ||||
| 23 | 6 | 1 | |||
| 24 | 2 | ||||
| 25 | 1 | ||||
| 26 | 2 | ||||
| 27 | 1 | ||||
| 28 | 3 | ||||
| 29 | 1 | ||||
| 30 | 2 | ||||
| 31 | 2 | ||||
| 32 | 6 | 6 | |||
| 33 | 7 | 1 | 2 | ||
| 34 | 2 | 7 | |||
| 35 | 2 | ||||
| 36 | 2 | ||||
| 37 | 1 | ||||
| 38 | 3 | ||||
| 40 | 1 | 1 | |||
| 41 | 7 | 3 | |||
| 42/45 | 7 | 1 | 1 | ||
| 43 | 1 | ||||
| 44 | 2 | 1 | |||
| 46 | 1 | ||||
| 47 | 5 | 3 | 1 | ||
| 48 | 1 | ||||
| 49 | 8 | 1 | 1 | ||
| 50 | 4 | ||||
| 51 | 2 | ||||
| 52 | 1 | ||||
| 53 | 4 | ||||
| 54 | 3 | ||||
| 55 | 1 | 1 | 1 | ||
| 56 | 2 | ||||
| 57 | 2 | ||||
| 58 | 1 | ||||
| 59 | 2 | ||||
| 60 | 3 | ||||
| 61 | 8 | 2 | 2 | 8 | 10 |
| 62 | 4 | ||||
| 63 | 7 | 2 | 1 | 1 | 1 |
| 64 | 6 | 1 | 3 | 1 | |
| 65 | 10 | 7 | 4 | 2 | 3 |
| 66 | 4 | 8 | 1 | ||
| 67 | 7 | 2 | 3 | 1 | 1 |
| 68 | 8 | 2 | 5 | 2 | |
P values are for significance level compared to control RMs.
Data are from reference 10.
In contrast, SVV transcription within ganglia of CD4-depleted RMs revealed that all SVV ORFs except ORF 39 were detected in at least one ganglion (Fig. 3 and Table 4). The breadth of transcription varied from animal to animal, with the detection of 64 of 69 ORFs in RM 24993 (Fig. 3B) and 15 of 69 ORFs in RM 25152 (Fig. 3D). Transcriptional analysis of ganglia from RM 24993 occurred at 63 d.p.i., and this animal exhibited a greater than 4-fold reduction in CD4+ T cells, whereas the ganglia of RM 25152 were harvested at 85 d.p.i., and the CD4+ T cells had recovered to predepletion levels (Table 1). Furthermore, the latent transcriptional profiles in ganglia from CD4-depleted RMs 24952 and 24993 were more similar to those obtained from bronchoalveolar lavage fluid cells (the site of inoculation) during acute infection (10), with the presence of numerous viral ORFs involved in DNA replication and virion assembly, although at reduced average copy numbers per μg of RNA (Table 4). Overall, the differences in the incidence of ORF expression between CD4-depleted and control RMs were statistically significant (P < 0.0001, Bonferroni-corrected P value of 0.0004) (Table 3). Moreover, the odds of ORF expression for CD4-depleted RMs were estimated to be 11.95 times those of control RMs (P = 0.0004), with a 95% confidence interval of 6.64 to 64.23. The incidence of SVV ORF expression remained significantly higher in the DRG-C, DRG-T, and DRG-L/S of CD4-depleted RMs than in the ganglia of control RMs (P < 0.0001, Bonferroni P value of 0.0004). Similarly, the odds ratio of SVV ORF expression remained significantly increased in CD4-depleted RMs compared to controls when specific ganglia were analyzed. The odds of SVV ORF expression in CD4-depleted RMs compared to control RMs were estimated to be (i) 12.37 times greater in DRG-C (95% confidence interval, 4.34 to 35.27), (ii) 27.36 times greater in DRG-T (95% confidence interval, 15.48 to 48.35), and (iii) 15.47 times greater in DRG-L/S (95% confidence interval, 6.97 to 34.35).
Fig 3.
SVV transcriptome in sensory ganglia of latently infected CD4-depleted animals. Total RNA was isolated from trigeminal ganglia (TG), cervical dorsal root ganglia (DRG-C), thoracic dorsal root ganglia (DRG-T), and lumbar/sacral dorsal root ganglia (DRG-L/S). RNA was amplified and analyzed by RT-qPCR using primer and probe sets specific for each viral ORF. Data shown are the average copy numbers for triplicate reactions per sample from 1 μg of total RNA.
Table 4.
Range of SVV gene expression in sensory ganglia for each RM
| ORF | Range of expression (avg no. of copies/μg total RNA)a in ganglia from indicated animal |
|||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CD4 depleted |
CD8 depleted |
CD20 depleted |
Aged |
|||||||||||
| 24952 | 24993 | 25111 | 25152 | 25343 | 25371 | 26108 | 26842 | 25833 | 25892 | 25905 | 25920 | 15449 | 15461 | |
| A | 2.22 × 103–1.26 × 105 | 2.38 × 104–1.45 × 103 | 4.84 × 102–2.77 × 104 | 8.12 × 102–7.18 × 103 | 0 | 1.65 × 102 | 5.88 × 102–6.91 × 103 | 1.65 × 103 | 1.00 × 103–1.84 × 103 | 0 | 1.68 × 102 | 0 | 0 | 1.52 × 105 |
| B | 4.37 × 102–1.08 × 104 | 4.94 × 102–6.75 × 104 | 3.68 × 103 | 1.27 × 103 | 0 | 1.28 × 102 | 2.36 × 102 | 0 | 1.52 × 102–2.18 × 102 | 0 | 0 | 0 | 0 | 1.25 × 102–1.65 × 102 |
| 1 | 1.61 × 102–3.13 × 103 | 3.74 × 102–1.44 × 104 | 0 | 0 | 0 | 1.78 × 102 | 0 | 0 | 0 | 1.37 × 102 | 0 | 0 | 2.16 × 102 | 1.54 × 103 |
| 3 | 2.54 × 102–9.24 × 102 | 6.83 × 102–2.22 × 103 | 4.89 × 102 | 0 | 0 | 1.25 × 102 | 0 | 0 | 0 | 0 | 0 | 2.40 × 102 | 0 | 2.25 × 102–2.84 × 102 |
| 4 | 2.69 × 104–4.56 × 104 | 1.48 × 104–2.10 × 106 | 1.00 × 102 | 0 | 0 | 1.75 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1.02 × 102–1.72 × 102 |
| 5 | 1.17 × 102–8.65 × 102 | 8.10 × 102–6.31 × 103 | 0 | 0 | 0 | 1.79 × 106 | 1.04 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 6 | 2.42 × 103 | 2.18 × 102–2.29 × 104 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 7 | 1.37 × 103–4.11 × 103 | 2.13 × 103–2.06 × 104 | 1.24 × 103 | 0 | 0 | 0 | 0 | 1.58 × 102–2.77 × 102 | 1.34 × 102–6.58 × 102 | 0 | 0 | 0 | 0 | 1.78 × 102 |
| 8 | 0 | 4.90 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1.21 × 102 | 0 |
| 9 | 3.87 × 102 | 1.29 × 103 | 3.47 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 9A | 3.24 × 102 | 1.32 × 102–3.95 × 103 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1.59 × 102–4.15 × 102 | 0 | 0 | 1.28 × 102 | 1.69 × 102 |
| 10 | 0 | 5.19 × 102–1.60 × 103 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 11 | 1.54 × 102–1.85 × 102 | 7.62 × 102–4.54 × 103 | 3.53 × 102 | 0 | 0 | 6.03 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1.06 × 102 |
| 12 | 0 | 0 | 4.13 × 102 | 0 | 0 | 1.01 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 13 | 1.23 × 103–1.24 × 103 | 3.30 × 102–5.09 × 103 | 1.40 × 104 | 6.50 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 4.08 × 102 | 0 |
| 14 | 3.13 × 102 | 2.48 × 102–8.11 × 102 | 4.68 × 102 | 0 | 0 | 1.04 × 102–3.75 × 103 | 1.48 × 103 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 15 | 1.11 × 103–1.37 × 103 | 1.88 × 102–4.55 × 103 | 3.14 × 103 | 1.91 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 3.37 × 102 | 9.57 × 103 |
| 16 | 1.22 × 102–4.06 × 102 | 7.20 × 102–2.07 × 103 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 17 | 3.36 × 102–4.19 × 102 | 2.80 × 102–1.13 × 103 | 1.72 × 103 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 18 | 6.66 × 102–1.11 × 103 | 2.37 × 102–7.84 × 103 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 3.02 × 102–3.58 × 102 | 0 |
| 19 | 7.82 × 102 | 4.52 × 103 | 0 | 0 | 0 | 0 | 2.09 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 20 | 1.08 × 102 | 1.48 × 102–6.95 × 102 | 2.39 × 104 | 0 | 0 | 1.11 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 21 | 1.29 × 103 | 4.98 × 103 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 22 | 0 | 0 | 6.06 × 102 | 3.24 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 23 | 1.67 × 103–9.52 × 103 | 2.33 × 102–6.12 × 104 | 1.92 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 6.94 × 102 | 0 |
| 24 | 0 | 1.04 × 103 | 8.57 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 25 | 0 | 4.32 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 26 | 0 | 5.64 × 102–9.76 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 27 | 0 | 3.56 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 28 | 2.33 × 102–1.55 × 103 | 1.09 × 103 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 29 | 0 | 5.29 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 30 | 0 | 1.25 × 102–5.76 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 31 | 3.11 × 102 | 1.86 × 103 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 32 | 2.51 × 102–1.29 × 103 | 2.55 × 102–3.64 × 103 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 5.00 × 102–8.34 × 102 | 1.46 × 102–2.73 × 103 |
| 33 | 1.26 × 102–1.67 × 103 | 1.43 × 102–1.89 × 103 | 0 | 1.67 × 102–1.92 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 1.03 × 102 | 0 | 1.51 × 102–2.65 × 102 | 0 |
| 34 | 0 | 3.49 × 102 | 0 | 2.02 × 102 | 1.87 × 102–3.28 × 102 | 2.14 × 102–5.62 × 102 | 3.11 × 102–2.41 × 103 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 35 | 0 | 8.21 × 102 | 1.90 × 103 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 36 | 0 | 2.94 × 102 | 3.28 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 37 | 0 | 3.78 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 38 | 5.29 × 102 | 2.79 × 102–6.56 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 39 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 40 | 0 | 2.16 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1.19 × 102 |
| 41 | 3.93 × 102–3.75 × 104 | 7.94 × 103–6.04 × 104 | 4.41 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1.28 × 102 | 2.02 × 102–1.10 × 104 |
| 42/45 | 1.85 × 102–5.64 × 102 | 2.15 × 102–1.98 × 103 | 2.47 × 103 | 2.92 × 102–6.42 × 102 | 0 | 0 | 4.82 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 1.89 × 102 |
| 43 | 0 | 2.03 × 103 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 44 | 0 | 2.22 × 102 | 1.08 × 102 | 0 | 0 | 7.79 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 46 | 0 | 2.91 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 47 | 1.64 × 102 | 8.64 × 102 | 0 | 1.06 × 102–3.35 × 103 | 0 | 1.53 × 102–2.87 × 103 | 8.32 × 102 | 0 | 0 | 2.08 × 103 | 0 | 0 | 0 | 0 |
| 48 | 0 | 0 | 1.75 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 49 | 1.50 × 103–6.09 × 103 | 2.98 × 102–2.31 × 104 | 2.52 × 104 | 6.5 × 103 | 0 | 0 | 4.66 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 2.69 × 102 |
| 50 | 1.72 × 102–3.77 × 102 | 1.04 × 102–7.18 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 51 | 1.06 × 102 | 3.63 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 52 | 0 | 1.83 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 53 | 5.42 × 102–2.47 × 103 | 7.91 × 103 | 6.00 × 103 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 54 | 2.80 × 102 | 3.52 × 102 | 5.59 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 55 | 0 | 2.01 × 103 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2.86 × 102 |
| 56 | 1.04 × 102 | 5.98 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 57 | 3.11 × 102 | 1.86 × 103 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 58 | 0 | 0 | 1.19 × 104 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 59 | 0 | 2.33 × 102 | 1.11 × 103 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 60 | 3.08 × 102–6.64 × 102 | 1.31 × 103 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 61 | 2.08 × 104–7.73 × 105 | 2.81 × 104–4.90 × 106 | 6.38 × 103 | 2.47 × 103 | 0 | 1.81 × 102 | 2.14 × 102 | 0 | 0 | 0 | 0 | 0 | 2.07 × 102–2.63 × 102 | 1.31 × 102–9.45 × 103 |
| 62 | 5.97 × 103 | 1.39 × 103–9.93 × 106 | 0 | 4.10 × 102 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 63 | 1.40 × 103–9.88 × 105 | 2.91 × 103–4.18 × 106 | 6.73 × 102 | 0 | 0 | 0 | 1.16 × 102–4.12 × 102 | 0 | 0 | 0 | 0 | 2.21 × 102 | 0 | 2.94 × 103 |
| 64 | 7.05 × 102–3.16 × 103 | 1.45 × 102–9.08 × 103 | 8.75 × 103 | 0 | 0 | 0 | 1.11 × 103 | 0 | 1.14 × 102–5.66 × 102 | 0 | 0 | 0 | 0 | 0 |
| 65 | 8.98 × 102–1.12 × 104 | 1.75 × 102–7.26 × 104 | 8.39 × 103 | 2.01 × 103–1.02 × 104 | 2.73 × 102–5.34 × 102 | 2.02 × 102 | 1.02 × 102–2.67 × 103 | 3.05 × 102 | 9.91 × 102–2.50 × 103 | 0 | 0 | 2.23 × 102–5.47 × 102 | 0 | 7.88 × 102–9.86 × 102 |
| 66 | 1.05 × 103 | 1.49 × 103–8.22 × 103 | 2.06 × 102 | 0 | 0 | 0 | 0 | 0 | 1.35 × 102–1.47 × 102 | 0 | 1.28 × 102–1.54 × 102 | 1.92 × 102–1.24 × 103 | 0 | 0 |
| 67 | 1.55 × 103–4.18 × 103 | 4.98 × 102–1.39 × 104 | 1.29 × 103 | 2.16 × 102 | 0 | 0 | 9.13 × 102–1.41 × 103 | 9.13 × 102 | 1.14 × 102–1.51 × 102 | 0 | 0 | 2.44 × 102 | 0 | 3.08 × 102 |
| 68 | 2.35 × 103–4.05 × 103 | 4.87 × 102–1.84 × 104 | 2.21 × 103 | 1.59 × 102–1.90 × 103 | 0 | 0 | 0 | 0 | 1.60 × 102–1.82 × 103 | 0 | 0 | 0 | 1.01 × 102–5.34 × 102 | 1.00 × 102 |
0, below limit of detection.
The breadth of SVV ORFs detected in the ganglia of CD8-depleted RMs also varied between animals, but the gene expression levels were notably reduced compared to the levels in CD4-depleted RMs (Table 4). Overall, we detected 22 of 69 ORFs in latent SVV transcriptional profiles from CD8-depleted RMs (Fig. 4 and Table 4), and the numbers of ORFs detected ranged from 2 in the sensory ganglia of RM 25343 (Fig. 4A) to 15 in the ganglia from RM 25371 (Fig. 4B). The additional transcripts detected compared to those in control nondepleted RMs mapped mainly within the core region of the genome and included SVV ORFs 5, 7, 11, 12, 14, 19, 20, 34, 42/45, 44, 47, and 49 (Table 3). SVV ORFs 5, 7, 11, 12, 14, 20, 44, and 49 are putative virion structural proteins, while SVV ORFs 19, 34, 42/45, and 47 have putative functions in viral replication (30). Overall, the incidence of SVV ORF expression in all ganglia from CD8-depleted RMs was not significantly different from that in control RMs (P = 0.07, Bonferroni P value of 1.0) (Table 3). However, the incidence of SVV transcription was significantly higher in TG of CD8-depleted RMs than in TG of control RMs (P = 0.0321). The odds of ORF expression in the TG were estimated to be 3.53 times greater for CD8-depleted RMs than for control RMs, with a 95% confidence interval of 1.11 to 11.23.
Fig 4.
SVV transcriptome in sensory ganglia of latently infected CD8-depleted animals. Total RNA was isolated from trigeminal ganglia (TG), cervical dorsal root ganglia (DRG-C), thoracic dorsal root ganglia (DRG-T), and lumbar/sacral dorsal root ganglia (DRG-L/S). RNA was amplified and analyzed by RT-qPCR using primer and probe sets specific for each viral ORF. Data shown are the average copy numbers for triplicate reactions per sample from 1 μg of total RNA.
The latent SVV transcriptional profiles in CD20-depleted RMs were comparable to those observed in nondepleted controls (Fig. 5 and Table 4). An additional 6 SVV transcripts (ORFs 1, 3, 7, 9A, 33, and 47) were detected in the CD20-depleted cohort that were not detected in control RMs (Table 3). The putative functions for these ORFs include virion assembly (ORFs 3 and 33), protein kinase (ORF 47), and virion structural proteins (ORFs 1, 7, and 9A) (30). Conversely, 3 transcripts (ORFs 4, 10, and 55) detected in control RMs were not detected in CD20-depleted RMs (Table 3). SVV ORFs 4 and 10 are transcriptional activators, while SVV ORF 55 is a putative component of the DNA helicase-primase complex (30).
Fig 5.
SVV transcriptome in sensory ganglia of latently infected CD20-depleted animals. Total RNA was isolated from trigeminal ganglia (TG), cervical dorsal root ganglia (DRG-C), thoracic dorsal root ganglia (DRG-T), and lumbar/sacral dorsal root ganglia (DRG-L/S). RNA was amplified and analyzed by RT-qPCR using primer and probe sets specific for each viral ORF. Data shown are the average copy numbers for triplicate reactions per sample from 1 μg of total RNA.
The impact of advanced age on SVV transcription in sensory ganglia.
Lastly, to assess the impact of age-related decline in immunity on SVV latency, we analyzed the SVV transcriptional profile from two aged rhesus macaques (19 years old) infected intrabronchially with 4 × 105 PFU WT SVV and euthanized at 64 d.p.i. The levels of SVV DNA in BAL fluid cells during acute infection were comparable between aged animals and juvenile control animals (Fig. 6A). In contrast, aged animals (15449 and 15461) experienced higher viral loads (P < 0.0001) in whole blood on days 35 and 42 postinfection, and RM 15461 also experienced higher viral loads (P < 0.0001) in whole blood on day 63 postinfection (Fig. 6B). Additionally, we measured the frequency of SVV-specific CD4 central memory and effector memory T cells by quantifying the total number of IFN-γ- and TNF-α-producing cells using intracellular cytokine staining after stimulation with SVV lysate (Fig. 6C and D). Our data show that aged RMs generated a delayed T cell response to SVV. Specifically, SVV-specific CD4 cells are detected at 7 and 14 d.p.i. in juvenile RMs but not until day 21 in aged RMs. The generation of SVV-specific IgG antibody titers was also delayed in aged RMs, particularly in RM 15461, which produced significantly reduced IgG titers at days 12 and 14 postinfection (Fig. 6E).
In sensory ganglia from the aged RMs, we detected SVV DNA only in the DRG-C (Table 2), in amounts of 40 and 23 copies per μg of DNA from aged RM 15449 and 15461, respectively. Absolute T cell and B cell numbers were also calculated for aged RMs preinfection and at necropsy and are detailed in Table 1. Transcriptional analysis of sensory ganglia from aged RMs detected 25 of 69 ORFs from portions of all 8 sensory ganglia (Fig. 7 and Table 4). Overall, aged RMs expressed SVV ORFs from throughout the SVV genome. Specifically, we detected 20 of 69 SVV ORFs in sensory ganglia from RM 15449 and 12 of 69 SVV ORFs in sensory ganglia from RM 15461 (Table 4). The most prevalent SVV ORFs detected in the aged RMs include ORF 32 (putative phosphoprotein), ORF 61 (transcriptional activator/latency-associated transcript), and ORF 68 (glycoprotein E) (Table 3) (30). The incidence of SVV ORF expression was significantly higher in aged RMs than in juvenile RMs (P < 0.0001) (Table 3). The odds of detecting SVV ORF expression were estimated to be 1.55 times greater in aged animals than in juvenile RMs (P = 0.0004), with a 95% confidence interval of 2.42 to 8.96. The increased incidence of SVV transcription in the TG (P < 0.0001, Bonferroni P value = 0.0004), DRG-C (P < 0.0001, Bonferroni P value = 0.0004), and DRG-L/S (P = 0.0038, Bonferroni P value = 0.0152) from aged RMs compared to those of juvenile RMs was also statistically significant.
Fig 7.
SVV transcriptome in sensory ganglia of latently infected aged animals. Total RNA was isolated from trigeminal ganglia (TG), cervical dorsal root ganglia (DRG-C), thoracic dorsal root ganglia (DRG-T), and lumbar/sacral dorsal root ganglia (DRG-L/S). RNA was amplified and analyzed by RT-qPCR using primer and probe sets specific for each viral ORF. Data shown are the average copy numbers for triplicate reactions per sample from 1 μg of total RNA.
DISCUSSION
The severity of primary VZV infection, as well as the incidence of VZV reactivation, increases in the immunocompromised and the elderly. More specifically, it is proposed that the loss of T cell-mediated immunity, whether due to age (immunosenescence), disease (HIV infection and hematological malignancies), or immunosuppressive treatments (chemotherapy and radiotherapy), plays a critical role in VZV pathogenesis. A large body of clinical data supports this hypothesis. For instance, HIV-positive children suffer from disseminated varicella and increased severity of associated morbidities, such as pneumonia and hepatitis (31). In contrast, VZV-specific antibodies are not crucial for recovery from primary infection (17, 32), and children suffering from agammaglobulinemia have uncomplicated varicella infections (11). Moreover, lymphoma patients and hematopoietic transplant recipients are at increased risk from HZ (33, 34). Finally, VZV antibody titers do not decrease with age (35), although, notably, the incidence of HZ increases with age. Taken together, these observations suggest that VZV-specific T cells play a more critical role in the resolution of primary infection and prevention of HZ than VZV-specific antibodies.
To further understand the impact of immune cell loss during primary infection on viral latency, we investigated the impact of T and B cell depletion and advanced age during primary infection of rhesus macaques on SVV gene expression during latency. SVV infection of rhesus macaques recapitulates many virological and immunological aspects of VZV infection of humans (24). In the present study, we used sensory ganglia collected from RMs in a previously described study investigating the role of T and B cells in the resolution of acute SVV infection (23). The results from that study demonstrated a critical role for CD4 T cells in the control of SVV replication during primary infection. The results from our current study show that CD4 T cells also play a large role in the establishment of latency in sensory ganglia. A recent study found that one of the risk factors for HZ in HIV-positive patients is a CD4 T cell count of less than 500 cells per microliter of blood (21). Of note, 3 CD4-depleted RMs in our study (24952, 24993, and 25111) (Table 1) had a cell count of approximately 200 CD4 T cells in peripheral blood mononuclear cells per microliter of whole blood at the time of necropsy. Interestingly, 24952 and 24993 displayed the highest levels of transcriptional activity in sensory ganglia (Fig. 3A and B). Collectively, the data strongly suggest a critical role for CD4 T cells in protection against primary infection and the establishment of latency.
Data from our previous study suggested that CD8 T cells play a limited role in the resolution of acute SVV infection. Data presented in this paper also suggest a limited role for CD8 T cells in the establishment of latency, since increased SVV transcription within the sensory ganglia of CD8-depleted RMs was only detected within the trigeminal ganglia (P = 0.0321). The numbers of ORFs detected in the DRGs were also higher than in the controls, but the difference was not statistically significant. It should be noted that, at necropsy, the CD8 T cell numbers within the CD8-depleted cohort were 1.2- to 3.5-fold lower than the baseline (Table 1). While CD8 T cells have been shown to be important in the control of reactivation of other alphaherpesviruses (i.e., HSV-1) (reviewed in reference 36), previous studies have shown that, in human sensory ganglia, activated CD8 T cells do not surround latently VZV-infected neurons (37). Similarly, after an episode of HZ, CD8 T cells were not found to be specifically surrounding VZV-positive neurons but, instead, were detected throughout the ganglion tissue tested (38). These data suggest that CD8 T cells may play a limited role in latency.
At the time of necropsy, the CD20+ cells had rebounded to preinfection levels (range, 0.8- to 1.5-fold change from predepletion to necropsy) (Table 1) in the CD20-depleted RMs, which also presented latent SVV transcriptional profiles similar to those observed in the control RMs (Fig. 7). These observations are consistent with the fact that the disease progression and severity during acute SVV infection in CD20-depleted RMs were similar to the progression and severity in nondepleted control RMs (23). Clinical studies also show that antibodies do not play a critical role in the control of acute or latent VZV infection (11, 35).
The development of age-related changes in the immune system of elderly rhesus macaques is similar to that described in humans, including a reduced immune response to infection and vaccination (reviewed in reference 39). To assess the impact of advanced age on viral latency, we compared SVV gene expression in sensory ganglia of two aged RMs 64 days after primary infection to that in juvenile controls. We detected a statistically significant increase in SVV gene expression during latency in aged animals compared to juvenile controls. The SVV ORFs detected in the aged RMs mapped to regions throughout the SVV genome. This observation could perhaps be due to decreased immune surveillance within the sensory ganglia of the aged RMs or, possibly, to the renewed viremia or the delayed T cell response to SVV observed in these aged animals (Fig. 6).
We have previously shown that the most frequently detected gene in juvenile control RMs during latency was SVV ORF 61 (10). SVV ORF 61 is a viral transactivator that also expresses an antisense transcript. ORF 61 sense transcripts are detected most often during acute infection, whereas antisense transcripts are more abundant during latent infection (40). ORF 61 was detected in 10 of 16 ganglia from control RMs, 8 of 8 ganglia from aged RMs, 8 of 12 ganglia from CD4-depleted RMs, 2 of 14 ganglia from CD8-depleted RMs, and 2 of 13 ganglia from CD20-depleted RMs (Table 3). The most prevalent SVV ORF expressed in control and aged RMs was ORF 61, but in CD4- and CD8-depleted RMs, ORF 65 was the most prevalent, and it was also detected during latency in control RMs in 3 of 16 ganglia. SVV ORF 65 is a putative tegument phosphoprotein, and the VZV homolog was shown to be unnecessary for viral replication both in cell culture and in vivo (41–43). In CD20-depleted RMs, ORF 66 was the most prevalent gene detected. ORF 66 was also detected during latency in control RMs in 2 of 16 ganglia. The VZV homolog of ORF 66 is a serine/threonine kinase that has been shown to phosphorylate ORF 62 (viral transactivator), preventing nuclear import (44–46), which provides a potential mechanism to help maintain viral latency. ORF 66 is also involved in downregulating major histocompatibility complex class I (47, 48), modulating IFN-γ in T cells (49), and inhibiting virus-induced apoptosis (49, 50).
In conclusion, our data show that the presence of cell-mediated immunity during the time of infection is important to the establishment of SVV latency and influences gene expression in sensory ganglia. Our data also suggest that the CD4 T cell population is crucial to the establishment and, possibly, the maintenance of latency, which we will investigate further in future studies.
ACKNOWLEDGMENTS
We thank Anj Stadnik, Kyung Park, and Erin Riscoe for technical assistance, the Division of Animal Resources (DAR) at the Oregon National Primate Research Center for expert animal care, especially Anne Lewis and Lois Colgin for conducting the necropsies and collecting tissues, and Alfred Legasse, Miranda Fischer, and Shannon Planer for collection of blood and BAL samples. We thank Daniel Streblow, Victor DeFilippis, and Ryan Estep for critical reading of the manuscript.
This work was supported by American Heart Association career development grant 0930234N, NIH grants R01AG037042, 2T32AI007472-16, and 8P51 OD011092-53, and the Brookdale Foundation.
Footnotes
Published ahead of print 22 May 2013
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