Presence of CD80 and Absence of LAT in Modulating Cellular Infiltration and HSV-1 Latency.

CD80 is the best-known costimulatory molecule for effective T cell functions. Many different reports have summarized the role of CD80 in HSV-1 and its functions in maintaining adaptive immunity, which is the main player in causing herpes stromal keratitis (HSK). To determine the effects of absence or overexpression of CD80 in HSV-1 infection, we infected CD80-/- and WT mice with a recombinant HSV-1 expressing murine CD80 (HSV-CD80) in place of the latency associated transcript (LAT). Parental dLAT2903 virus lacking LAT was used as a control. After infection, critical components of infection like virus replication, eye disease, early cellular infiltrates into the corneas and trigeminal ganglia (TG), latency-reactivation in the infected mice were determined. Our findings reveal that the absence of CD80 in the CD80-/- mice infected with both viruses did not affect the viral titers in the mice eyes or eye disease, but it played a significant role in critical components of HSV-induced immunopathology. The WT mice infected with dLAT2903 virus had significantly higher levels of latency compared with the CD80-/- mice infected with dLAT2903 virus, while levels of latency as determined by gB DNA expression were similar between the WT and CD80-/- mice infected with HSV-CD80 virus. In contrast to the differences in the levels of latency between the infected groups, the absence of CD80 expression in the CD80-/- mice or its overexpression by HSV-CD80 virus did not have any effect on the time of reactivation. Furthermore, the absence of CD80 expression contributed to more inflammation in the CD80-/--infected mice. Overall, this study suggests that in the absence of CD80, inflammation increases, latency is reduced, but reactivation is not affected. Altogether, our study suggests that reduced latency correlated with reduced levels of inflammatory molecules and blocking or reducing expression of CD80 could be used to mitigate the immune responses, therefore controlling HSV-induced infection.

A novel GFP-based strategy to quantitate cellular spatial associations in HSV-1 viral pathogenesis.

Periodic reactivation of herpes simplex virus type 1 (HSV-1) triggers immune responses that result in corneal scarring (CS), known as herpes stromal keratitis (HSK). Despite considerable research, fully understanding HSK and eliminating it remains challenging due to a lack of comprehensive analysis of HSV-1-infected immune cells in both corneas and trigeminal ganglia (TG). We engineered a recombinant HSV-1 expressing green fluorescent protein (GFP) in the virulent McKrae virus strain that does not require corneal scarification for efficient virus replication (GFP-McKrae). Next-generation sequencing (NGS) analysis, along with in vitro and in vivo assays, showed that GFP-McKrae virus was similar to WT-McKrae virus. Furthermore, corneal cells infected with GFP-McKrae were quantitatively analyzed using image mass cytometry (IMC). The single-cell reconstruction data generated cellular maps of corneas based on the expression of 25 immune cell markers in GFP-McKrae-infected mice. Corneas from mock control mice showed the presence of T cells and macrophages, whereas corneas from GFP-McKrae-infected mice on days 3 and 5 post-infection (PI) exhibited increased immune cells. Notably, on day 3 PI, increased GFP expression was observed in closely situated clusters of DCs, macrophages, and epithelial cells. By day 5 PI, macrophages and T cells became prominent. Finally, immunostaining methods detected HSV-1 or GFP and gD proteins in latently infected TG. This study presents a valuable strategy for identifying cellular spatial associations in viral pathogenesis and holds promise for future therapeutic applications.IMPORTANCEThe goal of this study was to establish quantitative approaches to analyze immune cell markers in HSV-1-infected intact corneas and trigeminal ganglia from primary and latently infected mice. This allowed us to define spatial and temporal interactions between specific immune cells and their potential roles in virus replication and latency. To accomplish this important goal, we took advantage of the utility of GFP-McKrae virus as a valuable research tool while also highlighting its potential to uncover previously unrecognized cell types that play pivotal roles in HSV-1 replication and latency. Such insights will pave the way for developing targeted therapeutic approaches to tackle HSV-1 infections more effectively.

A Quadruple Gene-Deleted Live BoHV-1 Subunit RVFV Vaccine Vector Reactivates from Latency and Replicates in the TG Neurons of Calves but Is Not Transported to and Shed from Nasal Mucosa.

Bovine herpesvirus type 1 (BoHV-1) establishes lifelong latency in trigeminal ganglionic (TG) neurons following intranasal and ocular infection in cattle. Periodically, the latent virus reactivates in the TG due to stress and is transported anterogradely to nerve endings in the nasal epithelium, where the virus replicates and sheds. Consequently, BoHV-1 is transmitted to susceptible animals and maintained in the cattle population. Modified live BoHV-1 vaccine strains (BoHV-1 MLV) also have a similar latency reactivation. Therefore, they circulate and are maintained in cattle herds. Additionally, they can regain virulence and cause vaccine outbreaks because they mutate and recombine with other circulating field wild-type (wt) strains. Recently, we constructed a BoHV-1 quadruple mutant virus (BoHV-1qmv) that lacks immune evasive properties due to UL49.5 and glycoprotein G (gG) deletions. In addition, it also lacks the gE cytoplasmic tail (gE CT) and Us9 gene sequences designed to make it safe, increase its vaccine efficacy against BoHV-1, and restrict its anterograde neuronal transport noted above. Further, we engineered the BoHV-1qmv-vector to serve as a subunit vaccine against the Rift Valley fever virus (BoHV-1qmv Sub-RVFV) (doi: 10.3390/v15112183). In this study, we determined the latency reactivation and nasal virus shedding properties of BoHV-1qmv (vector) and BoHV-1qmv-vectored subunit RVFV (BoHV-1qmv sub-RVFV) vaccine virus in calves in comparison to the BoHV-1 wild-type (wt) following intranasal inoculation. The real-time PCR results showed that BoHV-1 wt- but not the BoHV-1qmv vector- and BoHV-1qmv Sub-RVFV-inoculated calves shed virus in the nose following dexamethasone-induced latency reactivation; however, like the BoHV-1 wt, both the BoHV-1qmv vector and BoHV-1qmv Sub-RVFV viruses established latency, were reactivated, and replicated in the TG neurons. These results are consistent with the anterograde neurotransport function of the gE CT and Us9 sequences, which are deleted in the BoHV-1qmv and BoHV-1qmv Sub-RVFV.

Examination of neuro-inflammation and senescence in brainstem of aged mice latently infected with human alphaherpesvirus 1 (HSV-1).

Human alphaherpesvirus 1 (HSV-1) establishes life-long latency in sensory neurons in trigeminal ganglia (TG), brainstem neurons, and other CNS neurons. Two important segments of the brainstem were examined in this study: principal sensory nucleus of the spinal trigeminal tract (Pr5) because it receives direct afferent inputs from TG, and locus coeruleus (LC) because it is indirectly connected to Pr5 and LC sends axonal projections to cortical structures, which may facilitate viral spread from brainstem to the brain. The only viral gene abundantly expressed during latency is the latency associated transcript (LAT). Previous studies revealed 8-week old female C57Bl/6 mice infected with a LAT null mutant (dLAT2903) versus wild-type (wt) HSV-1 exhibit higher levels of senescence markers and inflammation in LC of females. New studies revealed 1-year old mice latently infected with wt HSV-1 or dLAT2903 contained differences in neuroinflammation and senescence in Pr5 and LC versus young mice. In summary, these studies confirm HSV-1 promotes neuro-inflammation in the brainstem, which may accelerate neurodegenerative disease.

A cell cycle regulator, E2F2, and glucocorticoid receptor cooperatively transactivate the bovine alphaherpesvirus 1 immediate early transcription unit 1 promoter.

Bovine alphaherpesvirus 1 (BoHV-1) infection causes respiratory tract disorders and immune suppression and may induce bacterial pneumonia. BoHV-1 establishes lifelong latency in sensory neurons after acute infection. Reactivation from latency consistently occurs following stress or intravenous injection of the synthetic corticosteroid dexamethasone (DEX), which mimics stress. The immediate early transcription unit 1 (IEtu1) promoter drives expression of infected cell protein 0 (bICP0) and bICP4, two viral transcriptional regulators necessary for productive infection and reactivation from latency. The IEtu1 promoter contains two glucocorticoid receptor (GR) responsive elements (GREs) that are transactivated by activated GR. GC-rich motifs, including consensus binding sites for specificity protein 1 (Sp1), are in the IEtu1 promoter sequences. E2F family members bind a consensus sequence (TTTCCCGC) and certain specificity protein 1 (Sp1) sites. Consequently, we hypothesized that certain E2F family members activate IEtu1 promoter activity. DEX treatment of latently infected calves increased the number of E2F2+ TG neurons. GR and E2F2, but not E2F1, E2F3a, or E2F3b, cooperatively transactivate a 436-bp cis-regulatory module in the IEtu1 promoter that contains both GREs. A luciferase reporter construct containing a 222-bp fragment downstream of the GREs was transactivated by E2F2 unless two adjacent Sp1 binding sites were mutated. Chromatin immunoprecipitation studies revealed that E2F2 occupied IEtu1 promoter sequences when the BoHV-1 genome was transfected into mouse neuroblastoma (Neuro-2A) or monkey kidney (CV-1) cells. In summary, these findings revealed that GR and E2F2 cooperatively transactivate IEtu1 promoter activity, which is predicted to influence the early stages of BoHV-1 reactivation from latency. IMPORTANCE: Bovine alpha-herpesvirus 1 (BoHV-1) acute infection in cattle leads to establishment of latency in sensory neurons in the trigeminal ganglia (TG). A synthetic corticosteroid dexamethasone consistently initiates BoHV-1 reactivation in latently infected calves. The BoHV-1 immediate early transcription unit 1 (IEtu1) promoter regulates expression of infected cell protein 0 (bICP0) and bICP4, two viral transcriptional regulators. Hence, the IEtu1 promoter must be activated for the reactivation to occur. The number of TG neurons expressing E2F2, a transcription factor and cell cycle regulator, increased during early stages of reactivation from latency. The glucocorticoid receptor (GR) and E2F2, but not E2F1, E2F3a, or E2F3b, cooperatively transactivated a 436-bp cis-regulatory module (CRM) in the IEtu1 promoter that contains two GR responsive elements. Chromatin immunoprecipitation studies revealed that E2F2 occupies IEtu1 promoter sequences in cultured cells. GR and E2F2 mediate cooperative transactivation of IEtu1 promoter activity, which is predicted to stimulate viral replication following stressful stimuli.

Herpes Simplex Virus Type 1 Preferentially Enhances Neuro-Inflammation and Senescence in Brainstem of Female Mice.

Following acute infection, herpes simplex virus 1 (HSV-1) establishes lifelong latency in neurons. The latency associated transcript (LAT) is the only viral gene abundantly expressed during latency. Wild-type (WT) HSV-1 reactivates more efficiently than LAT mutants because LAT promotes establishment and maintenance of latency. While sensory neurons in trigeminal ganglia (TG) are important sites for latency, brainstem is also a site for latency and reactivation from latency. The principal sensory nucleus of the spinal trigeminal tract (Pr5) likely harbors latent HSV-1 because it receives afferent inputs from TG. The locus coeruleus (LC), an adjacent brainstem region, sends axonal projections to cortical structures and is indirectly linked to Pr5. Senescent cells accumulate in the nervous system during aging and accelerate neurodegenerative processes. Generally senescent cells undergo irreversible cell cycle arrest and produce inflammatory cytokines and chemokines. Based on these observations, we hypothesized HSV-1 influences senescence and inflammation in Pr5 and LC of latently infected mice. This hypothesis was tested using a mouse model of infection. Strikingly, female but not age-matched male mice latently infected with a LAT null mutant (dLAT2903) exhibited significantly higher levels of senescence markers and inflammation in LC, including cell cycle inhibitor p16, NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3), IL-1α, and IL-ß. Conversely, Pr5 in female but not male mice latently infected with WT HSV-1 or dLAT2903 exhibited enhanced expression of important inflammatory markers. The predilection of HSV-1 to induce senescence and inflammation in key brainstem regions of female mice infers that enhanced neurodegeneration occurs. IMPORTANCE HSV-1 (herpes simplex virus 1), an important human pathogen, establishes lifelong latency in neurons in trigeminal ganglia and the central nervous system. In contrast to productive infection, the only viral transcript abundantly expressed in latently infected neurons is the latency associated transcript (LAT). The brainstem, including principal sensory nucleus of the spinal trigeminal tract (Pr5) and locus coeruleus (LC), may expedite HSV-1 spread from trigeminal ganglia to the brain. Enhanced senescence and expression of key inflammatory markers were detected in LC of female mice latently infected with a LAT null mutant (dLAT2903) relative to age-matched male or female mice latently infected with wild-type HSV-1. Conversely, wild-type HSV-1 and dLAT2903 induced higher levels of senescence and inflammatory markers in Pr5 of latently infected female mice. In summary, enhanced inflammation and senescence in LC and Pr5 of female mice latently infected with HSV-1 are predicted to accelerate neurodegeneration.

Antiviral CD19+CD27+ Memory B Cells Are Associated with Protection from Recurrent Asymptomatic Ocular Herpesvirus Infection.

Reactivation of herpes simplex virus 1 (HSV-1) from latently infected neurons of the trigeminal ganglia (TG) leads to blinding recurrent herpetic disease in symptomatic (SYMP) individuals. Although the role of T cells in herpes immunity seen in asymptomatic (ASYMP) individuals is heavily explored, the role of B cells is less investigated. In the present study, we evaluated whether B cells are associated with protective immunity against recurrent ocular herpes. The frequencies of circulating HSV-specific memory B cells and of memory follicular helper T cells (CD4+ Tfh cells), which help B cells produce antibodies, were compared between HSV-1-infected SYMP and ASYMP individuals. The levels of IgG/IgA and neutralizing antibodies were compared in SYMP and ASYMP individuals. We found that (i) the ASYMP individuals had increased frequencies of HSV-specific CD19+CD27+ memory B cells, and (ii) high frequencies of HSV-specific switched IgG+CD19+CD27+ memory B cells detected in ASYMP individuals were directly proportional to high frequencies of CD45R0+CXCR5+CD4+ memory Tfh cells. However, no differences were detected in the level of HSV-specific IgG/IgA antibodies in SYMP and ASYMP individuals. Using the UV-B-induced HSV-1 reactivation mouse model, we found increased frequencies of HSV-specific antibody-secreting plasma HSV-1 gD+CD138+ B cells within the TG and circulation of ASYMP mice compared to those of SYMP mice. In contrast, no significant differences in the frequencies of B cells were found in the cornea, spleen, and bone-marrow. Our findings suggest that circulating antibody-producing HSV-specific memory B cells recruited locally to the TG may contribute to protection from symptomatic recurrent ocular herpes. IMPORTANCE Reactivation of herpes simplex virus 1 (HSV-1) from latently infected neurons of the trigeminal ganglia (TG) leads to blinding recurrent herpetic disease in symptomatic (SYMP) individuals. Although the role of T cells in herpes immunity against blinding recurrent herpetic disease is heavily explored, the role of B cells is less investigated. In the present study, we found that in both asymptomatic (ASYMP) individuals and ASYMP mice, there were increased frequencies of HSV-specific memory B cells that were directly proportional to high frequencies of memory Tfh cells. Moreover, following UV-B-induced reactivation, we found increased frequencies of HSV-specific antibody-secreting plasma B cells within the TG and circulation of ASYMP mice compared to those of SYMP mice. Our findings suggest that circulating antibody-producing HSV-specific memory B cells recruited locally to the TG may contribute to protection from recurrent ocular herpes.

Headache as a Symptom of COVID-19: Narrative Review of 1-Year Research.

PURPOSE OF REVIEW: Headache is a common symptom of COVID-19 with emerging literature being published on the subject. Although it may seem unspecific, scientific evidence has allowed a better definition of this headache type, revealing relevant associations with other COVID-19 symptoms and prognoses. We therefore sought to highlight the most remarkable findings concerning headache secondary to COVID-19, specifically focusing on epidemiology, characteristics, pathophysiology, and treatments. RECENT FINDINGS: The real prevalence of headache as a symptom of COVID-19 is still unclear ranging from 10 to 70%. Headache mainly has a tension-type-like phenotype, although 25% of individuals present with migraine-like features that also occur in patients without personal migraine history. This finding suggests that a likely pathophysiological mechanism is the activation of the trigeminovascular system. SARS-CoV-2 neurotropism can occur by trans-synaptic invasion through the olfactory route from the nasal cavity, leading to anosmia which has been associated with headache. SARS-CoV-2 protein has been found not only in olfactory mucosa and bulbs but also in trigeminal branches and the trigeminal ganglion, supporting this hypothesis. However, other mechanisms such as brain vessels inflammation due to SARS-CoV-2 damage to the endothelium or systemic inflammation in the context of cytokine storm cannot be ruled out. Interestingly, headache has been associated with lower COVID-19 mortality. No specific treatment for COVID-19 headache is available at present. Studies show that investigating COVID-19 headache represents an opportunity not only to better understand COVID-19 in general but also to advance in the knowledge of both secondary and primary headaches. Future research is therefore warranted.

Local Immune Control of Latent Herpes Simplex Virus Type 1 in Ganglia of Mice and Man.

Herpes simplex virus type 1 (HSV-1) is a prevalent human pathogen. HSV-1 genomes persist in trigeminal ganglia neuronal nuclei as chromatinized episomes, while epithelial cells are typically killed by lytic infection. Fluctuations in anti-viral responses, broadly defined, may underlay periodic reactivations. The ganglionic immune response to HSV-1 infection includes cell-intrinsic responses in neurons, innate sensing by several cell types, and the infiltration and persistence of antigen-specific T-cells. The mechanisms specifying the contrasting fates of HSV-1 in neurons and epithelial cells may include differential genome silencing and chromatinization, dictated by variation in access of immune modulating viral tegument proteins to the cell body, and protection of neurons by autophagy. Innate responses have the capacity of recruiting additional immune cells and paracrine activity on parenchymal cells, for example via chemokines and type I interferons. In both mice and humans, HSV-1-specific CD8 and CD4 T-cells are recruited to ganglia, with mechanistic studies suggesting active roles in immune surveillance and control of reactivation. In this review we focus mainly on HSV-1 and the TG, comparing and contrasting where possible observational, interventional, and in vitro studies between humans and animal hosts.

Modulating glutamine metabolism to control viral immuno-inflammatory lesions.

Infection of the cornea with HSV results in an immune-inflammatory reaction orchestrated by proinflammatory T cells that is a major cause of human vision impairment. The severity of lesions can be reduced if the representation of inflammatory T cells is changed to increase the presence of T cells with regulatory function. This report shows that inhibiting glutamine metabolism using 6-Diazo-5-oxo-l-norleucine (DON) administered via intraperitoneal (IP) starting 6 days after ocular infection and continued until day 15 significantly reduced the severity of herpetic stromal keratitis lesions. The therapy resulted in reduced neutrophils, macrophages as well proinflammatory CD4 Th1 and Th17 T cells in the cornea, but had no effect on levels of regulatory T cells. A similar change in the representation of inflammatory and regulatory T cells occurred in the trigeminal ganglion (TG) the site where HSV infection establishes latency. Glutamine metabolism was shown to be required for the in-vitro optimal induction of both Th1 and Th17 T cells but not for the induction of Treg that were increased when glutamine metabolism was inhibited. Inhibiting glutamine metabolism also changed the ability of latently infected TG cells from animals previously infected with HSV to reactivate and produce infectious virus.

Absence of CD28-CTLA4-PD-L1 Costimulatory Molecules Reduces Herpes Simplex Virus 1 Reactivation.

We previously reported that herpes simplex virus 1 (HSV-1) ICP22 binds to CD80 and suppresses CD80 expression in vitro and in vivo. Similar to ICP22, the cellular costimulatory molecules CD28, CTLA4, and PD-L1 also bind to CD80. In this study, we asked whether, similar to ICP22-null virus, the absence of these costimulatory molecules will reduce HSV-1 infectivity. To test our hypothesis, CD28-/-, CD28-/- CTLA4-/-, PD-L1-/-, and wild-type control BALB/c mice were ocularly infected with HSV-1 strain KOS. Levels of virus replication in the eye, corneal scarring (CS), latency, and reactivation in infected mice were determined. Expression of different genes in the trigeminal ganglia (TG) of latently infected mice was also determined by NanoString and quantitative reverse transcription-PCR (qRT-PCR). In the absence of costimulatory molecules, latency levels were higher than those in wild-type control mice, but despite higher latency, a significant number of TG from infected knockout mice did not reactivate. Reduced reactivation correlated with downregulation of 26 similar cellular genes that are associated with inflammatory signaling and innate immune responses. These results suggest that lower reactivation directly correlates with lower expression of interferon signaling. Thus, despite having different modes of actions, we identified a similar function for CD28, CTLA4, and PD-L1 in HSV-1 reactivation that is dependent on their interactions with CD80. Therefore, blocking these interactions could be a therapeutic target for HSV-1-induced reactivation. IMPORTANCE Costimulatory molecules play an important role in activation of T cell responses, and T cells contribute to HSV-1-induced eye disease in the host. Similar to HSV-1 ICP22, the cellular costimulatory molecules CD28, CTLA4, and PD-L1 also bind to CD80. In this study, we have shown that the absence of these costimulatory molecules significantly reduced HSV-1 ex vivo reactivation. Therefore, inhibiting the binding of costimulatory molecules to CD80 could be used to reduce reactivation and, consequently, HSV-1-induced eye disease.

Herpes simplex virus-1 KOS-63 strain is virulent and causes titer-dependent corneal nerve damage and keratitis.

To investigate the acute clinical, immunological, and corneal nerve changes following corneal HSV-1 KOS-63 strain inoculation. Corneas of C57BL/6 mice were inoculated with either low dose (Ld) or high dose (Hd) HSV-1 KOS-63 or culture medium. Clinical evaluation was conducted up to 7 days post inoculation (dpi). Viral titers were assessed by standard plaque assay. Excised corneas were stained for CD45 and beta-III tubulin. Corneal flow cytometry was performed to assess changes in leukocyte subpopulations. Corneal sensation was measured using a Cochet-Bonnet esthesiometer. Naïve, sham-infected (post scarification), and McKrae-infected C57BL/6 corneas served as two negative and positive controls, respectively. Compared to Ld infected mice, Hd HSV-1 KOS-63 demonstrated higher incidence of corneal opacity (1.5 ×) and neovascularization (2.6 × ; p < 0.05). At 7 dpi Hd infected mice showed more severe corneal opacity (2.23 vs. 0.87; p = 0.0003), neovascularization (6.00 vs. 0.75; p < 0.0001), and blepharitis (3.11 vs. 2.06; p = 0.001) compared to the Ld group. At 3 dpi epitheliopathy was significantly larger in the Hd group (23.59% vs. 3.44%; p = 0.001). Similarly, corneal opacity was significantly higher in Hd McKrae-infected corneas as compared with Ld McKrae-infected corneas at 3 and 5 dpi. No significant corneal opacity, neovascularization, blepharitis, and epitheliopathy were observed in naïve or sham-infected mice. Higher viral titers were detected in corneas (1 and 3 dpi) and trigeminal ganglia (TG) (3 and 5 dpi) in Hd versus Ld KOS-63 groups (p < 0.05). Leukocyte density showed a gradual increase over time from 1 to 7 dpi in both KOS-63 and McKrae-infected corneas. Corneal flow cytometric analysis (3 dpi) demonstrated a higher percentage of Gr-1 + (71.6 vs. 26.3) and CD11b + (90.6 vs. 41.1) cells in Hd versus Ld KOS-63 groups. Corneal nerve density significantly decreased in both Hd KOS-63 and Hd McKrae infected corneas in comparison with naïve and sham-infected corneas. At 3 dpi corneal nerve density was lower in the Hd versus Ld KOS-63 groups (16.79 vs. 57.41 mm/mm2; p = 0.004). Corneal sensation decreased accordingly at 5 and 7 dpi in both Ld and Hd KOS-63-infected mice. Corneal inoculation with HSV-1 KOS-63 strain shows acute keratitis and nerve degeneration in a dose-dependent fashion, demonstrating virulence of this strain.

Inhibition of Stress-Induced Viral Promoters by a Bovine Herpesvirus 1 Non-Coding RNA and the Cellular Transcription Factor, ß-Catenin.

The ability to establish, maintain, and reactivate from latency in sensory neurons within trigeminal ganglia (TG) is crucial for bovine herpesvirus 1 (BoHV-1) transmission. In contrast to lytic infection, the only viral gene abundantly expressed during latency is the latency-related (LR) gene. The synthetic corticosteroid dexamethasone consistently induces reactivation from latency, in part because the glucocorticoid receptor (GR) transactivates viral promoters that drive expression of key viral transcriptional regulator proteins (bICP0 and bICP4). Within hours after dexamethasone treatment of latently infected calves, LR gene products and ß-catenin are not readily detected in TG neurons. Hence, we hypothesized that LR gene products and/or ß-catenin restrict GR-mediated transcriptional activation. A plasmid expressing LR RNA sequences that span open reading frame 2 (ORF2-Stop) inhibited GR-mediated transactivation of the BoHV-1 immediate early transcription unit 1 (IEtu1) and mouse mammary tumor virus (MMTV) promoter activity in mouse neuroblastoma cells (Neuro-2A). ORF2-Stop also reduced productive infection and GR steady-state protein levels in transfected Neuro-2A cells. Additional studies revealed that the constitutively active ß-catenin mutant reduced the transactivation of the IEtu1 promoter by GR and dexamethasone. Collectively, these studies suggest ORF2 RNA sequences and Wnt/ß-catenin signaling pathway actively promote maintenance of latency, in part, by impairing GR-mediated gene expression.

Varicella-zoster virus VLT-ORF63 fusion transcript induces broad viral gene expression during reactivation from neuronal latency.

Varicella-zoster virus (VZV) establishes lifelong neuronal latency in most humans world-wide, reactivating in one-third to cause herpes zoster and occasionally chronic pain. How VZV establishes, maintains and reactivates from latency is largely unknown. VZV transcription during latency is restricted to the latency-associated transcript (VLT) and RNA 63 (encoding ORF63) in naturally VZV-infected human trigeminal ganglia (TG). While significantly more abundant, VLT levels positively correlated with RNA 63 suggesting co-regulated transcription during latency. Here, we identify VLT-ORF63 fusion transcripts and confirm VLT-ORF63, but not RNA 63, expression in human TG neurons. During in vitro latency, VLT is transcribed, whereas VLT-ORF63 expression is induced by reactivation stimuli. One isoform of VLT-ORF63, encoding a fusion protein combining VLT and ORF63 proteins, induces broad viral gene transcription. Collectively, our findings show that VZV expresses a unique set of VLT-ORF63 transcripts, potentially involved in the transition from latency to lytic VZV infection.

Herpes Simplex Virus 1 (HSV-1) 0ΔNLS Live-Attenuated Vaccine Protects against Ocular HSV-1 Infection in the Absence of Neutralizing Antibody in HSV-1 gB T Cell Receptor-Specific Transgenic Mice.

The contribution of T cell and antibody responses following vaccination in resistance to herpes simplex virus 1 (HSV-1) infection continues to be rigorously investigated. In the present article, we explore the contribution of CD8+ T cells specific for the major antigenic epitope for HSV-1 glycoprotein B (gB498-505, gB) in C57BL/6 mice using a transgenic mouse (gBT-I.1) model vaccinated with HSV-1 0ΔNLS. gBT-I.1-vaccinated mice did not generate a robust neutralization antibody titer in comparison to the HSV-1 0ΔNLS-vaccinated wild-type C57BL/6 counterpart. Nevertheless, the vaccinated gBT-I.1 mice were resistant to ocular challenge with HSV-1 compared to vehicle-vaccinated animals based on survival and reduced corneal neovascularization but displayed similar levels of corneal opacity. Whereas there was no difference in the virus titer recovered from the cornea comparing vaccinated mice, HSV-1 0ΔNLS-vaccinated animals possessed significantly less infectious virus during acute infection in the trigeminal ganglia (TG) and brain stem compared to the control-vaccinated group. These results correlated with a significant increase in gB-elicited interferon-γ (IFN-γ), granzyme B, and CD107a and a reduction in lymphocyte activation gene 3 (LAG-3), programmed cell death 1 (PD-1), and T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) expressed by TG infiltrating gB-specific CD8+ T cells from the HSV-1 0ΔNLS-vaccinated group. Antibody depletion of CD8+ T cells in HSV-1 0ΔNLS-vaccinated mice rendered animals highly susceptible to virus-mediated mortality similar to control-vaccinated mice. Collectively, the HSV-1 0ΔNLS vaccine is effective against ocular HSV-1 challenge, reducing ocular neovascularization and suppressing peripheral nerve virus replication in the near absence of neutralizing antibody in this unique mouse model.IMPORTANCE The role of CD8+ T cells in antiviral efficacy using a live-attenuated virus as the vaccine is complicated by the humoral immune response. In the case of the herpes simplex virus 1 (HSV-1) 0ΔNLS vaccine, the correlate of protection has been defined to be primarily antibody driven. The current study shows that in the near absence of anti-HSV-1 antibody, vaccinated mice are protected from subsequent challenge with wild-type HSV-1 as measured by survival. The efficacy is lost following depletion of CD8+ T cells. Whereas increased survival and reduction in virus replication were observed in vaccinated mice challenged with HSV-1, cornea pathology was mixed with a reduction in neovascularization but no change in opacity. Collectively, the study suggests CD8+ T cells significantly contribute to the host adaptive immune response to HSV-1 challenge following vaccination with an attenuated virus, but multiple factors are involved in cornea pathology in response to ocular virus challenge.

Twelve Children with Varicella Vaccine Meningitis: Neuropathogenesis of Reactivated Live Attenuated Varicella Vaccine Virus.

Varicella vaccine is a live attenuated varicella-zoster virus (VZV). Like its parental strain called VZV pOka, the vaccine virus vOka retains some neurotropic properties. To better understand vOka neuropathogenesis, we reassessed 12 published cases of vOka meningitis that occurred in once-immunized and twice-immunized children, all of whom had bouts of herpes zoster preceding the central nervous system infection. Eight of the 12 meningitis cases occurred in children who had received only one immunization. There was no pattern to the time interval between varicella vaccination and the onset of herpes zoster with meningitis. Four of the meningitis cases occurred in children who had received two immunizations. Since all four children were 14 years old when meningitis was diagnosed, there was a strong pattern to the interval between the first vaccination at age 1 year and onset of meningitis, namely, 13 years. Knowledge of pathogenesis requires knowledge of the location of herpes zoster; the majority of dermatomal rashes occurred at sites of primary immunization on the arm or thigh, while herpes zoster ophthalmicus was uncommon. Based on this literature review, currently there is no consensus as to the cause of varicella vaccine meningitis in twice-immunized children.

Two Separate Tyrosine-Based YXXL/Φ Motifs within the Glycoprotein E Cytoplasmic Tail of Bovine Herpesvirus 1 Contribute in Virus Anterograde Neuronal Transport.

Bovine herpesvirus 1 (BHV-1) causes respiratory infection and abortion in cattle. Following a primary infection, BHV-1 establishes lifelong latency in the trigeminal ganglia (TG). Periodic reactivation of the latent virus in TG neurons results in anterograde virus transport to nerve endings in the nasal mucosa and nasal virus shedding. The BHV-1 glycoprotein E cytoplasmic tail (gE-CT) is necessary for virus cell-to-cell spread in epithelial cells and neuronal anterograde transport. Recently, we identified two tyrosine residues, Y467 and Y563, within the tyrosine-based motifs 467YTSL470 and 563YTVV566, which, together, account for the gE CT-mediated efficient cell-to-cell spread of BHV-1 in epithelial cells. Here, we determined that in primary neuron cultures in vitro, the individual alanine exchange Y467A or Y563A mutants had significantly diminished anterograde axonal spread. Remarkably, the double-alanine-exchanged Y467A/Y563A mutant virus was not transported anterogradely. Following intranasal infection of rabbits, both wild-type (wt) and the Y467A/Y563A mutant viruses established latency in the TG. Upon dexamethasone-induced reactivation, both wt and the mutant viruses reactivated and replicated equally efficiently in the TG. However, upon reactivation, only the wt, not the mutant, was isolated from nasal swabs. Therefore, the gE-CT tyrosine residues Y467 and Y563 together are required for gE CT-mediated anterograde neuronal transport.

RUNX Binding Sites Are Enriched in Herpesvirus Genomes, and RUNX1 Overexpression Leads to Herpes Simplex Virus 1 Suppression.

Herpes simplex virus 1 (HSV-1) and HSV-2 can efficiently establish lifelong, transcriptionally silent latency states in sensory neurons to escape host detection. While host factors have previously been associated with long-range insulators in the viral genome, it is still unknown whether host transcription factors can repress viral genes more proximately to promote latency in dorsal root ganglion (DRG) neurons. Here, we assessed whether RUNX (runt-related transcription factor) transcription factors, which are critical in the development of sensory neurons, could be binding HSV-1 genome directly to suppress viral gene expression and lytic infection. Using previously published transcriptome sequencing data, we confirmed that mouse DRG neurons highly express Runx1 mRNA. Through computational analysis of HSV-1 and HSV-2 genomes, we observed that putative RUNX consensus binding sites (CBSs) were more enriched and more closely located to viral gene transcription start sites than would be expected by chance. We further found that RUNX CBSs were significantly more enriched among genomes of herpesviruses compared to those of nonherpesviruses. Utilizing an in vitro model of HSV-1 infection, we found that overexpressed RUNX1 could bind putative binding sites in the HSV-1 genome, repress numerous viral genes spanning all three kinetic classes, and suppress productive infection. In contrast, knockdown of RUNX1 in neuroblastoma cells induced viral gene expression and increased HSV-1 infection in vitro In sum, these data support a novel role for RUNX1 in directly binding herpesvirus genome, silencing the transcription of numerous viral genes, and ultimately limiting overall infection.IMPORTANCE Infecting 90% of the global population, HSV-1 and HSV-2 represent some of the most prevalent viruses in the world. Much of their success can be attributed to their ability to establish lifelong latent infections in the dorsal root ganglia (DRG). It is still largely unknown, however, how host transcription factors are involved in establishing this latency. Here, we report that RUNX1, expressed highly in DRG, binds HSV-1 genome, represses transcription of numerous viral genes, and suppresses productive in vitro infection. Our computational work further suggests this strategy may be used by other herpesviruses to reinforce latency in a cell-specific manner.

Specific Akt Family Members Impair Stress-Mediated Transactivation of Viral Promoters and Enhance Neuronal Differentiation: Important Functions for Maintaining Latency.

Neurotropic Alphaherpesvirinae subfamily members such as bovine herpesvirus 1 (BoHV-1) and herpes simplex virus 1 (HSV-1) establish and maintain lifelong latent infections in neurons. Following infection of ocular, oral, or nasal cavities, sensory neurons within trigeminal ganglia (TG) are an important site for latency. Certain external stressors can trigger reactivation from latency, in part because activation of the glucocorticoid receptor (GR) stimulates productive infection and promoters that drive expression of key viral transcriptional regulators. The Akt serine/threonine protein kinase family is linked to maintaining latency. For example, Akt3 is detected in more TG neurons during BoHV-1 latency than in reactivation and uninfected calves. Furthermore, Akt signaling correlates with maintaining HSV-1 latency in certain neuronal models of latency. Finally, an active Akt protein kinase is crucial for the ability of the HSV-1 latency-associated transcript (LAT) to inhibit apoptosis in neuronal cell lines. Consequently, we hypothesized that viral and/or cellular factors impair stress-induced transcription and reduce the incidence of reactivation triggered by low levels of stress. New studies demonstrate that Akt1 and Akt2, but not Akt3, significantly reduced GR-mediated transactivation of the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter, the HSV-1 infected cell protein 0 (ICP0) promoter, and the mouse mammary tumor virus long terminal repeat (MMTV-LTR). Akt3, but not Akt1 or Akt2, significantly enhanced neurite formation in mouse neuroblastoma cells, which correlates with repairing damaged neurons. These studies suggest that unique biological properties of the three Akt family members promote the maintenance of latency in differentiated neurons.IMPORTANCE External stressful stimuli are known to increase the incidence of reactivation of Alphaherpesvirinae subfamily members. Activation of the glucocorticoid receptor (GR) by the synthetic corticosteroid dexamethasone (DEX) stimulates bovine herpesvirus 1 (BoHV-1) and herpes simplex virus 1 (HSV-1) reactivation. Furthermore, GR and dexamethasone stimulate productive infection and promoters that drive expression of viral transcriptional regulators. These observations lead us to predict that stress-induced transcription is impaired by factors abundantly expressed during latency. Interestingly, activation of the Akt family of serine/threonine protein kinases is linked to maintenance of latency. New studies reveal that Akt1 and Ak2, but not Akt3, impaired GR- and dexamethasone-mediated transactivation of the BoHV-1 immediate early transcription unit 1 and HSV-1 ICP0 promoters. Strikingly, Akt3, but not Akt1 or Akt2, stimulated neurite formation in mouse neuroblastoma cells, a requirement for neurogenesis. These studies provide insight into how Akt family members may promote the maintenance of lifelong latency.