Suppression of annexin A1 and its receptor reduces herpes simplex virus 1 lethality in mice.

Herpes simplex virus 1 (HSV-1)-induced encephalitis is the most common cause of sporadic, fatal encephalitis in humans. HSV-1 has at least 10 different envelope glycoproteins, which can promote virus infection. The ligands for most of the envelope glycoproteins and the significance of these ligands in virus-induced encephalitis remain elusive. Here, we show that glycoprotein E (gE) binds to the cellular protein, annexin A1 (Anx-A1) to enhance infection. Anx-A1 can be detected on the surface of cells permissive for HSV-1 before infection and on virions. Suppression of Anx-A1 or its receptor, formyl peptide receptor 2 (FPR2), on the cell surface and gE or Anx-A1 on HSV-1 envelopes reduced virus binding to cells. Importantly, Anx-A1 knockout, Anx-A1 knockdown, or treatments with the FPR2 antagonist reduced the mortality and tissue viral loads of infected mice. Our results show that Anx-A1 is a novel enhancing factor of HSV-1 infection. Anx-A1-deficient mice displayed no evident physiology and behavior changes. Hence, targeting Anx-A1 and FPR2 could be a promising prophylaxis or adjuvant therapy to decrease HSV-1 lethality.

Stroke propensity in the Th3+/ mouse model of ß-thalassemia intermedia.

ß-thalassemia is associated with multiple hematological and cerebrovascular symptoms linked to a hypercoagulable state that has not been fully replicated in animal models for the development of stroke treatments. Herein we compared the physiological properties and responses to transient cerebral hypoxia-ischemia (tHI) between six-month-old wildtype and heterozygous Th3/+ mice, a model of non-transfusion-dependent ß-thalassemia intermedia (ß-TI). We found that Th3/+ mice developed microcytic anemia, splenomegaly, higher platelet counts, and increased platelet-erythrocyte plus erythrocyte-leukocyte aggregates. Furthermore, Th3/+ mice showed diminished cerebrovascular reactivity (CVR) and cortical oxygen saturation under repetitive hypercapnic challenges. When subjected to a sub-threshold tHI insult, platelets and leukocytes in Th3/+ mice adhered to the cerebrovascular wall or formed aggregates, while their counterparts flew through smoothly in wildtype mice. Subsequently, Th3/+ mice showed increased fibrin deposition around cerebral blood vessels and larger infarction than wildtype mice, especially in female Th3/+ mice. Collectively these results showed that Th3/+ mice mimic key clinical features and a propensity to thromboembolism in ß-TI patients. The hypercoagulable state in Th3/+ mice is likely caused by multiple hematological and CVR anomalies that are similar, but are not identical to those in the mouse model of sickle cell anemia. As such, we suggest that Th3/+ mice are a useful model to study the pathological mechanisms and prophylactic stroke treatments in thalassemia patients.

Simultaneous determination of NO released inside and outside cells at the single-cell level using CE-LIF.

A simple, fast and reliable method based on capillary electrophoresis (CE) coupled with laser-induced fluorescence (LIF) detection was developed for the simultaneous analysis of NO released both inside and outside cells at the single-cell level closer to physiological conditions. After pre-capillary dual-labeling derivatization with a group of cells, single cells were injected into the separation capillary and lysed. The subsequent separation and detection of NO derivatives were achieved within 4.0 min producing mass limits of detection of 3.0 and 10.7 amol for intra- and extracellular NO, respectively. The developed method was successfully applied for simultaneous measurement of NO released both inside and outside single RAW 264.7 macrophage cells.

Simultaneous Quantitation of Intra- and Extracellular Nitric Oxide in Single Macrophage RAW 264.7 Cells by Capillary Electrophoresis with Laser-Induced Fluorescence Detection.

Single-cell analysis contributes to the understanding of cellular heterogeneity and behaviors. Nitric oxide (NO) is an important intracellular and intercellular signaling molecule, and the functions of NO are closely related to the balance between intra- and extracellular NO levels. In this manuscript, a convenient and reliable method based on a dual-labeling strategy using capillary electrophoresis (CE) separation with laser-induced fluorescence (LIF) detection has been presented for quantifying intra- and extracellular NO simultaneously in single cells. Followed by single-cell injection, a plug of HEPES buffer containing 1,3,5,7-tetramethyl-8-(3',4'-diaminophenyl)-difluoroboradiaza-s-indacene and disodium 2,6-disulfonate-1,3-dimethyl-5-hexadecyl-8-(3,4-diaminophenyl)-4,4'-difluoro-4-bora-3a,4a-diaza-s-indacene as the labeling reagents for intra- and extracellular NO, respectively, was aspirated from the inlet of the capillary. The on-line derivatization was carried out on the tip of the capillary at room temperature for 20 min. Then, the cell was lysed and NO derivatives were well separated within 14 min, producing mass detection limits (S/N = 3) of 2.4 and 8.1 amol for intra- and extracellular NO, respectively. The proposed method was validated by simultaneous analysis of intra- and extracellular NO in single macrophage cells. The dual labeling-based CE-LIF method holds great promise for research on the functions of NO as well as other bioactive molecules at the single-cell level.

Early neutrophil infiltration is critical for inflammation-sensitized hypoxic-ischemic brain injury in newborns.

Neutrophils are the most abundant leukocytes and usually the first immune cell-type recruited to a site of infection or tissue damage. In asphyxiated neonates, elevated peripheral neutrophil counts are associated with poorer neurological outcomes. Induced neutropenia provides brain protection in animal models of neonatal hypoxic-ischemic (HI) injury, but the anti-neutrophil serum used in past studies heavily cross-reacts with monocytes, thus complicating the interpretation of results. Here we examined neutrophil influx and extravasation, and used a specific anti-Ly6G antibody for induced neutropenia against lipopolysaccharide (LPS)-pretreated HI injury in murine neonates, a model for inflammation-sensitized hypoxic-ischemic encephalopathy (HIE). As early as 6 h after the LPS/HI insult, the mRNAs for neutrophil-recruiting and mitogenic chemokines ascended in the ipsilateral hemisphere, coinciding with immuno-detection of neutrophils. However, neutrophils mainly resided within blood vessels, exhibiting signs for neutrophil extracellular traps (NETs), before 48 h post-LPS/HI. Prophylactic anti-Ly6G treatment blocked the brain infiltration of neutrophils, but not monocytes or lymphocytes, and markedly decreased LPS/HI-induced pro-inflammatory cytokines, matrix metalloproteinase 9 (MMP-9), and brain tissue loss. In contrast, anti-Ly6G treatment at 4 h post-LPS/HI failed to prevent the influx of neutrophils and brain damage. Together, these results suggest important pathological functions for early-arriving neutrophils in inflammation-sensitized HIE.

Bortezomib induces HSV-1 lethality in mice with neutrophil deficiency.

Bortezomib suppressing NF-κB activity is an effective therapy for patients with myeloma or lymphoma. However, this drug can cause adverse effects, neutropenia, and recurrent infections of herpes viruses. Among herpes viruses, HSV-1 can reactivate to induce mortality. The important issues regarding how bortezomib diminishes neutrophils, whether bortezomib can induce HSV-1 reactivation, and how bortezomib exacerbates HSV-1 infection, need investigation. Using the murine model, we found that bortezomib induced HSV-1 reactivation. Bortezomib diminished neutrophil numbers in organs of uninfected and HSV-1-infected mice and turned a nonlethal infection to lethal with elevated tissue viral loads. In vitro results showed that bortezomib and HSV-1 collaborated to enhance the death and apoptosis of mouse neutrophils. The leukocyte deficiency induced by chemotherapies is generally believed to be the cause for aggravating virus infections. Here we show the potential of pathogen to exacerbate chemotherapy-induced leukocyte deficiency.

Effect of substituents on Stokes shift of BODIPY and its application in designing bioimaging probes.

BODIPY-based probes have excellent fluorescence properties. However, small Stokes shifts approximately 5-15 nm greatly affect their detection sensitivity. In this study, we compared the Stokes shifts of reported BODIPY-based probes with various of substituents, and found that the phenyl groups on the specific position of BODIPY core could expand the Stokes shift of BODIPY-based probes, and methoxy groups on these phenyl substituents could enhance such effects. Then, by quantum chemical calculations, we found that the number of methoxy groups might also have obvious effect on the Stokes shift of BODIPY. Taking nitric oxide (NO) as analyte, 4,4-difluoro-8-(3,4-diaminophenyl)-3,5-bis(2,4-dimethoxyphenyl)-4-bora-3a,4a-diaza-s-indancene (DMOPB) with diaminophenyl substituents has been designed and synthesized. Compared with monomethoxy-phenyl substituted BODIPY-based probes (MOPBs) in our previous work, Stokes shift of DMOPB was expanded by 10 nm when using dimethoxyphenyl instead of monomethoxyphenyl, which is basically consistent with the quantum chemistry calculation of 11 nm. DMOPB can react with NO in only 2 min to form the triazole DMOPB-T with a fluorescence quantum yield of 0.32. An excellent linear relationship was observed in the range of NO concentration from 0.5 µM to 4 µM and the detection limit was 1 nM. The experimental results indicate that DMOPB with high sensitivity, excellent selectivity, low toxicity and dark background can be a great candidate for imaging NO in cells and tissues. Considering the lack of practical way to increase Stokes shift of small-molecule fluorescent probes based on specific fluorophore, the proposed strategy has great potential for the designing of probes with large Stokes shift.

Suppression of interleukin-6 increases enterovirus A71 lethality in mice.

BACKGROUND: Enterovirus A71 (EV-A71) infection can induce fatal encephalitis in young children. Clinical reports show that interleukin-6 (IL-6) levels in the serum and cerebrospinal fluid of infected patients with brainstem encephalitis are significantly elevated. We used a murine model to address the significance of endogenous IL-6 in EV-A71 infection. RESULTS: EV-A71 infection transiently increased serum and brain IL-6 protein levels in mice. Most importantly, absence of IL-6 due to gene knockout or depletion of IL-6 using neutralizing monoclonal antibody enhanced the mortality and tissue viral load of infected mice. Absence of IL-6 increased the damage in the central nervous system and decreased the lymphocyte and virus-specific antibody responses of infected mice. CONCLUSIONS: Endogenous IL-6 functions to clear virus and protect the host from EV-A71 infection. Our study raises caution over the use of anti-IL-6 antibody or pentoxifylline to reduce IL-6 for patient treatment.

A cell surface specific two-photon fluorescent probe for monitoring intercellular transmission of hydrogen sulfide.

Hydrogen sulfide (H2S) is a new endogenously generated gasotransmitter and has implicated in many physiologies and pathologies closely related to its intracellular and intercellular signaling transduction. Although many fluorescent probes have been exploited to track and quantify H2S in living systems, none of them could be used for monitoring intercellular transmission of H2S. Herein, we developed a cell surface specific H2S probe, 4-azido-6-sulfo-N-hexadecyl-1,8-naphthalimide, sodium salt (ASNHN-N3), trying to investigate the behaviors of extracellular release of H2S. ASNHN-N3 is week fluorescent and could react with H2S at 37 °C in pH 7.4 buffer solutions to form product ASNHN-NH2 with strong fluorescence (Φ = 0.22). Using ASNHN-N3 as H2S probe, excellent linear correlation versus the concentration of NaHS was obtained ranging from 0 to 10 µM and the detection limit was 0.75 µM. With the lipid anchor and the hydrophilic sulfonic group introduced into the 1,8-naphthalimide (a skeleton of two-photon fluorescent probe), the amphiphilic probe is located at the surface of living cells which can record H2S efflux from the cell diffusing across the plasma membrane in living cells and deep-tissue by using two-photon microscopy. Thus we present a new strategy for further studying the mechanism of signaling molecules in cell communication and signal pathways.

Simultaneous monitoring of intra- and extracellular nitric oxide in living cells by means of dual-color fluorescence imaging.

A dual-color fluorescence imaging method for simultaneous monitoring of intra- and extracellular nitric oxide (NO) was developed. Assisted by confocal laser scanning microscope, the intra- and extracellular NO can be successfully visualized by using two selected probes, 4,4-difluoro-8-(3,4-diaminophenyl)-3,5-bis(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene (p-MOPB) and disodium 2,6-disulfonate-1,3-dimethyl-5-hexadecyl-8-(3,4-diaminophenyl)-4,4'-difluoro-4-bora-3a,4a-diaza-s-indacene (DSDMHDAB), which display distinct membrane permeability and show different colors of fluorescence after reaction with NO. Results indicated that intra- and extracellular NO could be fluorometrically detected without mutual interference. The applicability of the proposed method was validated by dual-color imaging of NO on both sides of the plasma membrane in RAW 264.7 murine macrophages and human vascular endothelial (ECV-304) cells. This multi-labeling approach using multi-laser excitation and multi-color fluorescence detection holds great promise for simultaneous analysis of NO as well as other gasotransmitters in living cells with subcellular resolution.

Thymidine Kinase-Negative Herpes Simplex Virus 1 Can Efficiently Establish Persistent Infection in Neural Tissues of Nude Mice.

Herpes simplex virus 1 (HSV-1) establishes latency in neural tissues of immunocompetent mice but persists in both peripheral and neural tissues of lymphocyte-deficient mice. Thymidine kinase (TK) is believed to be essential for HSV-1 to persist in neural tissues of immunocompromised mice, because infectious virus of a mutant with defects in both TK and UL24 is detected only in peripheral tissues, but not in neural tissues, of severe combined immunodeficiency mice (T. Valyi-Nagy, R. M. Gesser, B. Raengsakulrach, S. L. Deshmane, B. P. Randazzo, A. J. Dillner, and N. W. Fraser, Virology 199:484-490, 1994, https://doi.org/10.1006/viro.1994.1150). Here we find infiltration of CD4 and CD8 T cells in peripheral and neural tissues of mice infected with a TK-negative mutant. We therefore investigated the significance of viral TK and host T cells for HSV-1 to persist in neural tissues using three genetically engineered mutants with defects in only TK or in both TK and UL24 and two strains of nude mice. Surprisingly, all three mutants establish persistent infection in up to 100% of brain stems and 93% of trigeminal ganglia of adult nude mice at 28 days postinfection, as measured by the recovery of infectious virus. Thus, in mouse neural tissues, host T cells block persistent HSV-1 infection, and viral TK is dispensable for the virus to establish persistent infection. Furthermore, we found 30- to 200-fold more virus in neural tissues than in the eye and detected glycoprotein C, a true late viral antigen, in brainstem neurons of nude mice persistently infected with the TK-negative mutant, suggesting that adult mouse neurons can support the replication of TK-negative HSV-1. IMPORTANCE: Acyclovir is used to treat herpes simplex virus 1 (HSV-1)-infected immunocompromised patients, but treatment is hindered by the emergence of drug-resistant viruses, mostly those with mutations in viral thymidine kinase (TK), which activates acyclovir. TK mutants are detected in brains of immunocompromised patients with persistent infection. However, answers to the questions as to whether TK-negative (TK-) HSV-1 can establish persistent infection in brains of immunocompromised hosts and whether neurons in vivo are permissive for TK- HSV-1 remain elusive. Using three genetically engineered HSV-1 TK- mutants and two strains of nude mice deficient in T cells, we found that all three HSV-1 TK- mutants can efficiently establish persistent infection in the brain stem and trigeminal ganglion and detected glycoprotein C, a true late viral antigen, in brainstem neurons. Our study provides evidence that TK- HSV-1 can persist in neural tissues and replicate in brain neurons of immunocompromised hosts.

An Amphiphilic Fluorescent Probe Designed for Extracellular Visualization of Nitric Oxide Released from Living Cells.

Nitric oxide (NO) is an intracellular and intercellular messenger involved in numerous physiological and pathophysiological processes. Small-molecule fluorescent probes coupled with fluorescence microscopy provide excellent tools for real-time detection of NO in situ. However, most probes are designed for imaging intracellular NO, which cannot reflect the release behavior of endogenously produced NO. In order to visualize extracellular NO released from living cells, we report herein a particularly designed amphiphilic fluorescent probe, disodium 2,6-disulfonate-1,3-dimethyl-5-hexadecyl-8-(3,4-diaminophenyl)-4,4'-difluoro-4-bora-3a,4a-diaza-s-indacene (DSDMHDAB), in which hydrophilic groups are introduced to keep the fluorophore and recognition domain outside the cell and a hydrophobic C16 alkyl chain acts as the membrane anchor. Based on this design, NO released out of the cells has been visualized on the outer surface of the plasma membrane. Using RAW 264.7 cells and ECV-304 cells as models, the diffusion of NO across the plasma membrane has been directly observed. The amphiphilic design strategy of fluorescent probes holds great promise for developing fluorescent imaging probes to study the release behaviors of other endogenous gasotransmitters.

In vivo reactivation of latent herpes simplex virus 1 in mice can occur in the brain before occurring in the trigeminal ganglion.

UNLABELLED: Herpes simplex virus 1 (HSV-1) establishes latency in neurons of the brains and sensory ganglia of humans and experimentally infected mice. The latent virus can reactivate to cause recurrent infection. Both primary and recurrent infections can induce diseases, such as encephalitis. In humans, the majority of encephalitis cases occur as a recurrent infection. However, in the past, numerous mouse studies documented that viral reactivation occurs efficiently in the ganglion, but extremely rarely in the brain, when assessed ex vivo by cultivating minced tissue explants. Here, we compare the brains and the trigeminal ganglia of mice latently infected with HSV-1 (strain 294.1 or McKrae) for levels of viral genomes and in vivo reactivation. The numbers of copies of 294.1 and McKrae genomes in the brain stem were significantly greater than those in the trigeminal ganglion. Most importantly, 294.1 and McKrae reactivation was detected in the brain stems earlier than in the trigeminal ganglia of mice treated with hyperthermia to reactivate latent virus in vivo. In addition, the brain stem yielded reactivated virus at a high frequency compared with the trigeminal ganglion, especially in mice latently infected with 294.1 after hyperthermia treatment. These results provide evidence that recurrent brain infection can be induced by the reactivation of latent virus in the brain in situ. IMPORTANCE: Herpes simplex virus 1 (HSV-1) establishes latency in neurons of the brains and sensory ganglia of humans and experimentally infected mice. The latent virus can reactivate to cause recurrent infection. In the past, studies of viral reactivation focused on the ganglion, because efficient viral reactivation was detected in the ganglion but not in the brain when assessed ex vivo by cultivating mouse tissue explants. In this study, we report that the brain contains more viral genomes than the trigeminal ganglion in latently infected mice. Notably, the brain yields reactivated virus early and efficiently compared with the trigeminal ganglion after mice are stimulated to reactivate latent virus. Our findings raise the potential importance of HSV-1 latent infection and reactivation in the brain.

Tranylcypromine reduces herpes simplex virus 1 infection in mice.

Herpes simplex virus 1 (HSV-1) infects the majority of the human population and establishes latency by maintaining viral genomes in neurons of sensory ganglia. Latent virus can undergo reactivation to cause recurrent infection. Both primary and recurrent infections can cause devastating diseases, including encephalitis and corneal blindness. Acyclovir is used to treat patients, but virus resistance to acyclovir is frequently reported. Recent in vitro findings reveal that pretreatment of cells with tranylcypromine (TCP), a drug widely used in the clinic to treat neurological disorders, restrains HSV-1 gene transcription by inhibiting the histone-modifying enzyme lysine-specific demethylase 1. The present study was designed to examine the anti-HSV-1 efficacy of TCP in vivo because of the paucity of reports on this issue. Using the murine model, we found that TCP decreased the severity of wild-type-virus-induced encephalitis and corneal blindness, infection with the acyclovir-resistant (thymidine kinase-negative) HSV-1 mutant, and tissue viral loads. Additionally, TCP blocked in vivo viral reactivation in trigeminal ganglia. These results support the therapeutic potential of TCP for controlling HSV-1 infection.

Factors affecting herpes simplex virus reactivation from the explanted mouse brain.

The majority of encephalitis induced by herpes simplex virus type I (HSV-1) is due to viral reactivation from latency, but few studies have investigated the factors influencing viral reactivation in the brain due to the lack of a sensitive assay. We have established an ex vivo explant assay, which induced efficient viral reactivation in the dissociated mouse brain. Applying this assay, we investigated the infection of four HSV-1 strains with varying degrees of neurovirulence in three mouse strains with different levels of susceptibility to HSV-1 infection. We found that virulent HSV-1 strains and susceptible mouse strains exhibited prolonged viral growth during acute infection, increased latent viral genomes, and efficient explant reactivation in the brain stem. Collectively, both viral neurovirulence and host susceptibility positively correlate with HSV-1 reactivation from the explanted mouse brain.

Suppression of transcription factor early growth response 1 reduces herpes simplex virus 1-induced corneal disease in mice.

Herpes simplex virus 1 replication initiates angiogenesis and inflammation in the cornea. This can result in herpetic stromal keratitis (HSK), which is a leading cause of infection-induced corneal blindness. Host cellular factors mediate the progression of HSK, but little is known about these cellular factors and their mechanisms of action. We show here that the expression of the cellular transcription factor early growth response 1 (Egr-1) in HSV-1-infected mouse corneas was enhanced. Enhanced Egr-1 expression aggravated HSK by increasing viral replication and subsequent neovascularization with high levels of potent angiogenic factors, fibroblast growth factor 2, and vascular endothelial growth factor. Furthermore, Egr-1 deficiency due to a targeted disruption of the gene or knockdown of Egr-1 expression topically using a DNA-based enzyme significantly reduced HSK by decreasing both viral replication and the angiogenic response. The present study provides the first evidence that endogenous Egr-1 aggravates HSK and that blocking Egr-1 reduces corneal damage.

Beta interferon plus gamma interferon efficiently reduces acyclovir-resistant herpes simplex virus infection in mice in a T-cell-independent manner.

Acyclovir (ACV)-resistant herpes simplex virus type 1 (HSV-1) causes severe diseases in immunocompromised patients, so identification of new therapies is needed. Interferons (IFNs) are used to treat several other viral infections in the clinic, and IFN-beta and IFN-gamma are known to cooperatively reduce wild-type HSV-1 replication in the corneas of immunocompetent mice. Because IFN-gamma has been shown to exert an antiviral effect mostly through T cells, whether combined IFN treatment can still inhibit ACV-resistant HSV-1 replication, especially in immunocompromised hosts, is unknown. The present study evaluated the efficacy of combined IFN treatment on ACV-resistant HSV-1 mutants. In vitro results showed that IFN-beta acted synergistically with IFN-gamma to inhibit HSV-1 replication in both human and mouse cell lines. Some ACV-resistant mutants were actually hypersensitive to combined IFN treatment. In vivo results showed that topical treatment with a low dose of IFN-beta plus IFN-gamma (200 U each) on mouse corneas efficiently reduced the viral loads by up to 4, 4 and 3 logs, respectively, in the eyes, trigeminal ganglia and brainstems of wild-type and also immunocompromised nude mice infected or co-infected with ACV-resistant HSV-1 in a manner independent of T cells. A highly efficient reduction in HSV acute replication by combined IFN treatment led to a dramatic decrease in subsequent virus reactivation from neural tissues, trigeminal ganglia, brainstems and spinal cords of latently infected mice. Thus, a combination of IFN-beta and IFN-gamma could be a potential treatment for ACV-resistant HSV-1 in immunocompromised patients.

Suppression of transcription factor early growth response 1 reduces herpes simplex virus lethality in mice.

Herpes simplex virus type 1 (HSV-1) infection is the most common cause of sporadic, fatal encephalitis, but current understanding of how the virus interacts with cellular factors to regulate disease progression is limited. Here, we show that HSV-1 infection induced the expression of the cellular transcription factor early growth response 1 (Egr-1) in a human neuronal cell line. Egr-1 increased viral replication by activating promoters of viral productive cycle genes through binding to its corresponding sequences in the viral promoters. Mouse studies confirmed that Egr-1 expression was enhanced in HSV-1-infected brains and that Egr-1 functions to promote viral replication in embryonic fibroblasts. Furthermore, Egr-1 deficiency or knockdown of Egr-1 by a DNA-based enzyme greatly reduced the mortality of HSV-1-infected mice by decreasing viral loads in tissues. This study provides what we believe is the first evidence that Egr-1 increases the mortality of HSV-1 encephalitis by enhancing viral replication. Moreover, blocking this cellular machinery exploited by the virus could prevent host mortality.

Efficient reactivation of latent herpes simplex virus from mouse central nervous system tissues.

For decades, numerous ex vivo studies have documented that latent herpes simplex virus (HSV) reactivates efficiently from ganglia, but rarely from the central nervous systems (CNS), of mice when assayed by mincing tissues before explant culture, despite the presence of viral genomes in both sites. Here we show that 88% of mouse brain stems reactivated latent virus when they were dissociated into cell suspensions before ex vivo explant culture. The efficient reactivation of HSV from the mouse CNS was demonstrated with more than one viral strain, viral serotype, and mouse strain, further indicating that the CNS can be an authentic latency site for HSV with the potential to cause recurrent disease.