Reactivation of Herpes Simplex Virus Type 1 in the Mouse Trigeminal Ganglion: an In Vivo Study of Virus Antigen and Cytokines

Reactivation of herpes simplex virus type 1 (HSV-1) in the trigeminal ganglion (TG) was induced by UV irradiation of the corneas of latently infected mice. Immunocytochemistry was used to monitor the dynamics of cytokine (interleukin-2 [IL-2], IL-4, IL-6, IL-10, gamma interferon [IFN-γ], and tumor necrosis factor alpha [TNF-α]) and viral antigen production in the TG and the adjacent central nervous system on days 1 to 4, 6, 7, and 10 after irradiation. UV irradiation induced increased expression of IL-6 and TNF-α from satellite cells in uninfected TG. In latently infected TG, prior to reactivation, all satellite cells were TNF-α⁺ and most were also IL-6⁺. Reactivation, evidenced by HSV-1 antigens and/or infiltrating immune cells, occurred in 28 of 45 (62%) TG samples. Viral antigens were present in the TG in neurons, often disintegrating on days 2 to 6 after irradiation. Infected neurons were usually surrounded by satellite cells and the foci of immune cells producing TNF-α and/or IL-6. IL-4⁺ cells were detected as early as day 3 and were more numerous by day 10 (a very few IL-2⁺ and/or IFN-γ⁺ cells were seen at this time). No IL-10 was detected at any time. Our observations indicate that UV irradiation of the cornea may modulate cytokine production by satellite cells. We confirm that neurons are the site of reactivation and that they probably do not survive this event. The predominance of TNF-α and IL-6 following reactivation parallels primary infection in the TG and suggests a role in viral clearance. The presence of Th2-type cytokines (IL-4 and IL-6) indicates a role for antibody. Thus, several clearance mechanisms may be at work.     

Establishment of HSV1 Latency in Immunodeficient Mice Facilitates Efficient In Vivo Reactivation

The establishment of latent infections in sensory neurons is a remarkably effective immune evasion strategy that accounts for the widespread dissemination of life long Herpes Simplex Virus type 1 (HSV1) infections in humans. Periodic reactivation of latent virus results in asymptomatic shedding and transmission of HSV1 or recurrent disease that is usually mild but can be severe. An in-depth understanding of the mechanisms regulating the maintenance of latency and reactivation are essential for developing new approaches to block reactivation. However, the lack of a reliable mouse model that supports efficient in vivo reactivation (IVR) resulting in production of infectious HSV1 and/or disease has hampered progress. Since HSV1 reactivation is enhanced in immunosuppressed hosts, we exploited the antiviral and immunomodulatory activities of IVIG (intravenous immunoglobulins) to promote survival of latently infected immunodeficient Rag mice. Latently infected Rag mice derived by high dose (HD), but not low dose (LD), HSV1 inoculation exhibited spontaneous reactivation. Following hyperthermia stress (HS), the majority of HD inoculated mice developed HSV1 encephalitis (HSE) rapidly and synchronously, whereas for LD inoculated mice reactivated HSV1 persisted only transiently in trigeminal ganglia (Tg). T cells, but not B cells, were required to suppress spontaneous reactivation in HD inoculated latently infected mice. Transfer of HSV1 memory but not OVA specific or naïve T cells prior to HS blocked IVR, revealing the utility of this powerful Rag latency model for studying immune mechanisms involved in control of reactivation. Crossing Rag mice to various knockout strains and infecting them with wild type or mutant HSV1 strains is expected to provide novel insights into the role of specific cellular and viral genes in reactivation, thereby facilitating identification of new targets with the potential to block reactivation.     

Comprehensive quantification of herpes simplex virus latency at the single-cell level.

To date, characterization of latently infected tissue with respect to the number of cells in the tissue harboring the viral genome and the number of viral genomes contained within individual latently infected cells has not been possible. This level of cellular quantification is a critical step in determining (i) viral or host cell factors which function in the establishment and maintenance of latency, (ii) the relationship between latency burden and reactivation, and (iii) the effectiveness of vaccines or antivirals in reducing or preventing the establishment of latent infections. Presented here is a novel approach for the quantitative analysis of nucleic acids within the individual cells comprising complex solid tissues. One unique feature is that the analysis reflects the nucleic acids within the individual cells as they were in the context of the intact tissue-hence the name CXA, for contextual analysis. Trigeminal ganglia latently infected with herpes simplex virus (HSV) were analyzed by CXA of viral DNA. Both the type and the number of cells harboring the viral genome as well as the number of viral genomes within the individual latently infected cells were determined. Here it is demonstrated that (i) the long-term repository of HSV-1 DNA in the ganglion is the neuron, (ii) the viral-genome copy number within individual latently infected neurons is variable, ranging over 3 orders of magnitude from <10 to >1,000, (iii) there is a direct correlation between increasing viral input titer and the number of neurons in which latency is established in the ganglion, (iv) increasing viral input titer results in more neurons with greater numbers of viral-genome copies, (v) treatment with acyclovir (ACV) during acute infection reduces the number of latently infected ganglionic neurons 20-fold, and (vi) ACV treatment results in uniformly low (<10)-copy-number latency. This report represents the first comprehensive quantification of HSV latency at the level of single cells. Beyond viral latency, CXA has the potential to advance many studies in which rare cellular events occur in the background of a complex solid tissue mass, including microbial pathogenesis, tumorigenesis, and analysis of gene transfer.     

Medroxyprogesterone acetate inhibits CD8+ T cell viral-specific effector function and induces herpes simplex virus type 1 reactivation

Clinical research suggests hormonal contraceptive use is associated with increased frequencies of HSV reactivation and shedding. We examined the effects of medroxyprogesterone acetate (MPA), the compound most commonly used for injectable hormonal contraception, on HSV type 1 (HSV-1) reactivation and CD8(+) T cell function in murine trigeminal ganglia (TG). In ex vivo TG cultures, MPA dramatically inhibited canonical CD8(+) T cell effector functions, including IFN-gamma production and lytic granule release, and increased HSV-1 reactivation from latency. In vivo, MPA treatment of latently infected ovariectomized mice inhibited IFN-gamma production and lytic granule release by TG resident CD8(+) T cells stimulated directly ex vivo. RNA specific for the essential immediate early viral gene ICP4 as well as viral genome DNA copy number were increased in mice that received MPA during latency, suggesting that treatment increased in vivo reactivation. The increase in HSV-1 copy number appeared to be the result of a two-tine effect, as MPA induced higher reactivation frequencies from latently infected explanted TG neurons in the presence or absence of CD45(+) cells. Our data suggest hormonal contraceptives that contain MPA may promote increased frequency of HSV reactivation from latency through the combinatory effects of inhibiting protective CD8(+) T cell responses and by a leukocyte-independent effect on infected neurons.     

Lack of Interleukin-6 (IL-6) Enhances Susceptibility to Infection but Does Not Alter Latency or Reactivation of Herpes Simplex Virus Type 1 in IL-6 Knockout Mice

The ability of the pleotropic, proinflammatory cytokine interleukin-6 (IL-6) to affect the replication, latency, and reactivation of herpes simplex virus type 1 (HSV-1) in cell culture and in IL-6 knockout (KO) mice was studied. In initial studies, we found no effect of exogenous IL-6, monoclonal antibodies to IL-6, or monoclonal antibody to the IL-6 coreceptor, gp130, on HSV-1 replication in vitro by plaque assay or reactivation ex vivo by explant cocultivation of latently infected murine trigeminal ganglia (TG). Compared with the wild-type (WT) mice, the IL-6 KO mice were less able to survive an ocular challenge with 10(5) PFU of HSV-1 (McKrae) (40% survival of WT and 7% survival KO mice; P = 0.01). There was a sixfold higher 50% lethal dose of HSV-1 in WT than IL-6 KO mice (1.7 x 10(4) and 2.7 x 10(3) PFU, respectively). No differences were observed in titers of virus recovered from the eyes, TG, or brains or in the rates of virus reactivation by explant cocultivation of TG from latently infected WT or KO mice. Exposure of latently infected mice to UV light resulted in comparable rates of reactivation and in the proportions of WT and KO animals experiencing reactivation. Moreover, quantitative PCR assays showed nearly identical numbers of HSV-1 genomes in latently infected WT and IL-6 KO mice. These studies indicate that while IL-6 plays a role in the protection of mice from lethal HSV infection, it does not substantively influence HSV replication, spread to the nervous system, establishment of latency, or reactivation.     

Signaling through Toll-Like Receptors Induces Murine Gammaherpesvirus 68 Reactivation In Vivo

Murine gammaherpesvirus 68 (MHV68) establishes a lifelong infection in mice and is used as a model pathogen to study the role of viral and host factors in chronic infection. The maintenance of chronic MHV68 infection, at least in some latency reservoirs, appears to be dependent on the capacity of the virus to reactivate from latency in vivo. However, the signals that lead to MHV68 reactivation in vivo are not well characterized. Toll-like receptors (TLRs), by recognizing the specific patterns of microbial components, play an essential role in the activation of innate immunity. In the present study, we investigated the capacity of TLR ligands to induce MHV68 reactivation, both in vitro and in vivo. The stimulation of latently infected B cell lines with ligands for TLRs 3, 4, 5, and 9 enhanced MHV68 reactivation; the ex vivo stimulation of latently infected primary splenocytes, recovered from infected mice, with poly(I:C), lipopolysaccharide, flagellin, or CpG DNA led to early B-cell activation, B-cell proliferation, and a significant increase in the frequency of latently infected cells reactivating the virus. In vivo TLR stimulation also induced B-cell activation and MHV68 reactivation, resulting in heightened levels of virus replication in the lungs which correlated with an increase in MHV68-specific CD8⁺ T-cell responses. Importantly, TLR stimulation also led to an increase in MHV68 latency, as evidenced by an increase in viral genome-positive cells 2 weeks post-in vivo stimulation by specific TLR ligands. Thus, these data demonstrate that TLR stimulation can drive MHV68 reactivation from latency and suggests that periodic pathogen exposure may contribute to the homeostatic maintenance of chronic gammaherpesvirus infection through stimulating virus reactivation and reseeding latency reservoirs.     

Rates of reactivation of latent herpes simplex virus from mouse trigeminal ganglia ex vivo correlate directly with viral load and inversely with number of infiltrating CD8+ T cells

Herpes simplex viruses (HSV) reactivate at rates proportional to the viral loads in latently infected ganglia. However, these rates vary substantially among infected animals. We assessed whether the numbers of HSV-specific CD8(+) T cells infiltrating latently infected ganglia also affect reactivation rates and contribute to their variability. Following corneal infection of mice with HSV type 2 (HSV-2), we quantified the latent viral loads in dissociated trigeminal ganglia by real-time PCR, the numbers of infiltrating CD8(+) T cells by flow cytometry, and the rates of reactivation by the detection of cell-free virus released from ganglion cells cultured in 96-well plates. The reactivation rates correlated directly with the latent viral loads (P = 0.001) but did so more strongly (P = 10(-7)) when cultures were depleted of CD8(+) T cells. Reactivation rates were reduced in a dose-dependent fashion by adding back ganglion CD8(+) T cells to the cultures (P = 0.003). We related the latent viral loads, numbers of CD8(+) T cells, and reactivation rates by mathematical equations. The rates of reactivation predicted from latent viral loads and numbers of infiltrating CD8(+) T cells in dissociated ganglia correlated with the observed rates of reactivation (P = 0.04). The reactivation of HSV-2 from ganglia ex vivo is determined both by the latent viral load and the number of infiltrating CD8(+) T cells.     

Quantitative polymerase chain reaction analysis of herpes simplex virus DNA in ganglia of mice infected with replication-incompetent mutants.

To study the roles of viral genes in the establishment and maintenance of herpes simplex virus (HSV) latency, we have developed a polymerase chain reaction assay that is both quantitative and sensitive. Using this assay, we analyzed the levels of viral DNA in trigeminal ganglia of mice inoculated corneally with HSV mutants that are defective for virus replication at one or more sites in mice and for reactivation upon ganglionic explant. Ganglia from mice infected with thymidine kinase-negative mutants, which replicate at the site of inoculation and establish latency but do not replicate acutely in ganglia or reactivate upon explant, contained a range of levels of HSV DNA that overlapped with the range found in ganglia latently infected with wild-type virus. On average, these mutant-infected ganglia contained one copy of HSV DNA per 100 cell equivalents (ca. 10(4) molecules), which was 50-fold less than the average for wild-type virus. Ganglia from mice infected with a ribonucleotide reductase deletion mutant, which is defective for acute replication and reactivation upon ganglionic explant, also contained on average one copy of HSV DNA per 100 cell equivalents. We also detected substantial numbers of HSV DNA molecules (up to ca. 10(3] in ganglia of mice infected with an ICP4 deletion mutant and other replication-negative mutants that are severely impaired for viral DNA replication and gene expression. These results raise the possibility that such mutants can establish latency, which could have important implications for mechanisms of latency and for vaccine and antiviral drug development.     

Shortening of the Symptom-Free Period in Rhesus Macaques Is Associated with Decreasing Nonsynonymous Variation in the env Variable Regions of Simian Immunodeficiency Virus SIVsm during Passage

During six blood passages of simian immunodeficiency virus SIVsm in rhesus macaques, the asymptomatic period shortened from 18 months to 1 month. To study SIVsm envelope gene (env) evolution during passage in rhesus macaques, the C1 to CD4 binding regions of multiple clones were sequenced at seroconversion and again at death. The env variation found during adaptation was almost completely confined to the variable regions. Intrasample sequence variation among clones at seroconversion was lower than the variation among clones at death. Intrasample variation among clones from a single time point as well as intersample variation decreased during the passage. In the variable regions, the mean number of intrasample nonsynonymous nucleotide substitutions decreased from the first passage (5.26 × 10⁻² ± 0.6 × 10⁻² per site) to the fifth passage (2.24 × 10⁻² ± 0.4 × 10⁻² per site), whereas in the constant regions, the mean number of intrasample nonsynonymous nucleotide substitutions differed less between the first and fifth passages (1.14 × 10⁻² ± 0.27 × 10⁻² and 0.80 × 10⁻² ± 0.24 × 10⁻² per site). Shortening of the asymptomatic period coincided with a rise in the Ks/Ka ratio (ratio between the number of synonymous [Ks] and the number of nonsynonymous [Ka] substitutions) from 1.080 in passage one to 1.428 in passage five and mimicked the difference seen in the intrahost evolution between asymptomatic and fast-progressing individuals infected with human immunodeficiency virus type 1. The distribution of nonsynonymous substitutions was biphasic, with most of the adaptation of env variable regions occurring in the first three passages. This phase, in which the symptom-free period fell to 4 months, was followed by a plateau phase of apparently reduced adaptation. Analysis of codon usage revealed decreased codon redundancy in the variable regions. Overall, the results suggested a biphasic pattern of adaptation and evolution, with extremely rapid selection in the first three passages followed by an equilibrium or stabilization of the variation between env clones at different time points in passages four to six.     

Herpes simplex virus type 1 promoter activity during latency establishment, maintenance, and reactivation in primary dorsal root neurons in vitro

A neonatal rat dorsal root ganglion-derived neuronal culture system has been utilized to study herpes simplex virus (HSV) latency establishment, maintenance, and reactivation. We present our initial characterization of viral gene expression in neurons following infection with replication-defective HSV recombinants carrying beta-galactosidase and/or green fluorescent protein reporter genes under the control of lytic cycle- or latency-associated promoters. In this system lytic virus reporter promoter activity was detected in up to 58% of neurons 24 h after infection. Lytic cycle reporter promoters were shut down over time, and long-term survival of neurons harboring latent virus genomes was demonstrated. Latency-associated promoter-driven reporter gene expression was detected in neurons from early times postinfection and was stably maintained in up to 83% of neurons for at least 3 weeks. In latently infected cultures, silent lytic cycle promoters could be activated in up to 53% of neurons by nerve growth factor withdrawal or through inhibition of histone deacetylases by trichostatin A. We conclude that the use of recombinant viruses containing reporter genes, under the regulation of lytic and latency promoter control in neuronal cultures in which latency can be established and reactivation can be induced, is a potentially powerful system in which to study the molecular events that occur during HSV infection of neurons.