Problem: Primary HSV-2 infection during pregnancy is associated with adverse pregnancy outcomes. However the mechanisms underlying these outcomes remain largely unknown. In this study we developed and characterized a mouse model of primary HSV-2 infection during early pregnancy and examined its effects on pregnancy and fetal outcomes. Methods of Study: C57BL/6 female mice positive for vaginal plugs were infected intravaginally (IVAG) with 10^3/10^4/10^5 PFU/mouse of HSV-2 (333) or saline (control) on gestational day (GD) 5. For comparison, female mice in diestrus stage were infected with HSV-2 at the same doses. Survival, pathology scores and vaginal viral shedding were measured post-infection. Systemic viral dissemination was examined by real-time PCR. Vaginal tissue, implantation sites, placenta and fetuses were examined by histology. Maternal serum (GD 13) and amniotic fluid (GD 8) was collected for multiplex cytokine analysis. Results: The minimum viral inoculation dose for infection in pregnant mice was 10^3 PFU of HSV-2, compared to 100-fold higher dose required to infect diestrus mice (10^5 PFU). There was a dose-dependent increase in implantation failure and number of resorptions with increasing dose of viral inoculum in pregnant mice at GD 8. In the 10^3 PFU group, although vaginal viral shedding was observed in all mice, 75% survived the infection, while all the mice in 10^4 and 10^5 PFU groups succumbed to infection by GD 13-15. There was evidence of abnormal placental morphology and necrotic fetal tissues in HSV-2 infected, pregnant mice compared to controls. Presence of HSV-2 DNA was measured in the vaginal tract, uterus (mated non-pregnant mice), and implantations of infected mated mice. HSV-2 DNA was also present in the spleen of the GD 13 time point group. Conclusions: These results indicate a 100-fold increase in susceptibility to HSV-2 infection during early pregnancy. At higher inoculation doses, IVAG HSV-2 infection spread systemically resulting in poor pregnancy outcomes and maternal mortality, especially in later gestation. At lower inoculation dose, the infection was localized in the reproductive tract and implantation sites, resulting in increased inflammation and adverse outcomes. This model will help to understand pathological mechanisms underlying adverse outcomes following primary HSV-2 infection in pregnancy. / Thesis / Master of Science (MSc)

Unlike RNA viruses, which typically encode their own RNA-dependent RNA polymerases, DNA viruses typically utilize host RNA Polymerase II (RNAPII) to transcribe their genes. Therefore, host factors that interact with RNAPII often maintain important regulatory roles during DNA virus infections. Senataxin (SETX) is a ubiquitously expressed 303 kDa RNA:DNA helicase that associates with RNAPII. It is involved in the resolution of R-loops and plays a role during the DNA damage response. Mutations in SETX are implicated in the neurodegenerative diseases Type 2 Ataxia with Oculomotor Apraxia (AOA2) and juvenile Amyotrophic Lateral Sclerosis (ALS4). Recent work from our group has demonstrated that SETX also acts as an antagonist of the antiviral response during RNA virus infections. Infections, including those caused by Herpes Simplex Virus type I (HSV-1), have been identified as potential environmental triggers of neurodegenerative diseases. Therefore, we elected to study the role of SETX during DNA virus infections since, in addition to regulating host genes, it may also play a role in viral transcription and/or DNA replication. Our data suggests that SETX is involved in the regulation of viral gene expression, and that SETX facilitates DNA replication and contributes to viral biogenesis. SETX attenuates the antiviral response, and in mouse models of infection, is protective against HSV-1 disease pathogenesis. These studies have enhanced our understanding of the role played by SETX during viral infection and may shed light on the mechanism(s) through which SETX dysfunction results in neurodegenerative diseases. / Thesis / Master of Health Sciences (MSc) / DNA viruses utilize host proteins in gene expression, therefore, associated factors play roles during DNA virus infections. Senataxin (SETX) is a RNA:DNA helicase associated with these proteins. SETX mutations are implicated in the neurodegenerative diseases Type 2 Ataxia with Oculomotor Apraxia (AOA2) and juvenile Amyotrophic Lateral Sclerosis (ALS4). Recently, our group demonstrated SETX antagonizes antiviral responses to RNA virus infections. Infections, including those caused by Herpes Simplex Virus type I (HSV-1), are identified as potential triggers of neurodegenerative diseases. We elected to study the role of SETX during DNA virus infections. Our data suggests that SETX is involved in the regulation of viral gene expression, facilitates HSV-1 DNA replication, attenuates the antiviral response, and in mouse models of infection, is protective against HSV-1 disease pathogenesis. These studies enhance understanding of the role of SETX during viral infection and may shed light on the mechanism(s) of SETX role in neurodegenerative disease.

Herpes simplex virus is an important model in the study of temporally regulated gene expression in eukaryotic cells. Three classes of genes - immediate early, early, and late - are sequentially expressed during the course of lytic infection. One immediate early gene product, ICP4, is required for transactivation of most early and late genes; it is also implicated in repression of immediate early gene expression. ICP4's mechanism(s) of action is/are not yet understood; although ICP4 binds to specific sequences of DNA, whether this is necessary for transregulation by ICP4 is not clear. To gain a better understanding of how the ability of ICP4 to bind DNA relates to its transregulatory activities, I introduced ICP4 binding sites into a simple model promoter within the viral genome. Two sets of construct were made in which an ICP4 binding site (or mutant site) was placed either downstream or upstream of a TATA box, reproducing the spacing found in (i) the native 𝘐𝘊𝘗4 promoter and (ii) the native 𝘐𝘊𝘗0 promoter (respectively). The promoter of HSV-1 𝘜𝘓24𝘣 (a nonessential gene in tissue culture) was replaced with these model promoters and levels of transcripts accumulating from these constructs during lytic infection assayed by primer extension. I found that an ICP4 site placed either upstream or downstream of a TATA box shifted kinetics of expression from E/leaky L to true L. Neither the strength of the TATA box nor the helical orientation of the ICP4 binding site with respect to the TATA box affected this result. / Thesis / Master of Science (MS)

Problem: Herpes simplex virus type-2 (HSV-2) is one of the most prevalent sexually transmitted infections in the world, and rates of infection are higher in women compared to men. Furthermore, vaccines developed against HSV-2 have failed at various stages of clinical trials, due to their inability to induce protective mucosal immunity. In animal models, intranasal (IN) immunization with attenuated HSV-2 (TK−) virus has been shown to confer protection against wildtype HSV-2 challenge. Since IN immunization serves as a more practical and less intrusive vaccination strategy, further studies are warranted to characterize optimal immune responses following IN immunization. We have previously demonstrated that estradiol (E2) treatment promotes enhanced protection against HSV-2 through enhanced anti-viral T cells responses. However, the effect of E2 on B cell responses, which were recently shown to be critical in protecting the host following IN immunization, remain poorly understood. Therefore, in this study we aimed to examine if following IN immunization, E2 enhances the memory B cell (MBC) and antibody-secreting plasma cell populations within the secondary lymphoid tissues and nasal effector sites, and whether this enhancement leads to an overall better protection against intravaginal IVAG WT-HSV-2 challenge. Methodology: Ovariectomized (OVX) mouse model of HSV-2 were pre-treated with E2 or placebo pellets. Subsequently, both groups were immunized intranasally with TK- HSV-2. Four weeks later nasal associated lymphoid tissues, nasal mucosa, cervical and iliac lymph nodes, spleen and vaginal tract were collected and processed and MBC and antibody-secreting plasma cells were characterized by flow cytometric analysis. HSV-2 specific IgM and IgG antibody responses in serum and vaginal secretions were measured by ELISA. In parallel experiments, animals were IVAG challenged with WT-HSV-2 and the B cell subsets were characterized as above. Results: The formation of MBC subsets, as seen by the presence of CD19+ IgD- cells and the heterogenous expression of CD73, CD80, and PD-L2, were observed four-weeks post immunization within the cervical and iliac lymph nodes and spleen, which were further enhanced in the presence of E2. Additionally, E2-treated mice had increased number of B220- CD138+ IgG2c+ plasma cells within the nasal mucosa following immunization. These enhancements translated into increased levels of HSV-2 specific IgG2b and IgG2c antibodies within the serum and vaginal secretions of E2-treated mice at four-weeks post IN immunization. Upon IVAG challenge, E2-treated mice, but not control mice, were protected. Since the antibody isotypes that were enhanced in E2 treated mice are correlated with Th17 responses, E2 mediated antibody enhancement was tested in IL-17 knockout mice. E2 treatment in IL-17-knockout mice failed to induce similar responses observed in WT mice, indicating that the enhancement of B cells and antibodies seen following E2 treatment was mediated in an IL-17 dependent manner. Conclusion: This study highlights the importance of sex-dependent differences in vaccine-induced immunity. Specifically, the findings from this study will provide valuable information for the design of a potentially efficacious mucosal vaccine strategy, whereby immunization in the context of E2 could significantly enhance antigen-specific antibody responses in the genital tract. / Thesis / Master of Science (MSc)

The prevalence of herpes simplex virus (HSV) infection globally is high, and although there is no cure for it, the antiviral drug acyclovir is used to alleviate symptoms. There are two types of HSV: HSV-1, which typically infects the oral area, and HSV-2, which is associated with genital infections. A mother who carries the infection may transmit it to a neonate in different ways, most commonly via vaginal delivery in the presence of active lesions. There are three types of HSV disease that affect newborns: skin, eyes or mouth (SEM) disease, central nervous system (CNS) disease, or disseminated disease. The purpose of this study was to examine the long-term effects of the infection and the treatment used in neonates infected with HSV. Data collection consisted of original case reports published in Medline, CINHAL, and Google Scholar. Two case reports were found, and this narrative review compares the cases, which report recurrences and outcomes of HSV infection identified in the three databases. Both cases were consistent with recurrence of CNS disease, and one showed signs of a slight developmental delay that may have been related to the CNS insult.

No description available.

No description available.

Herpes simplex virus entry into cells is a multistep process that engages the host cell machinery. The proteasome is a large, ATP-dependent, multisubunit protease that plays a critical role in the maintenance of cell homeostasis. A battery of assays were used to demonstrate that proteasome inhibitors blocked an early step in herpes simplex virus entry that occurred after capsid penetration into the cytosol but prior to capsid arrival at the nuclear periphery. Proteasome-dependent viral entry was not reliant on host or viral protein synthesis. MG132, a peptide aldehyde that competitively inhibits the degradative activity of the proteasome, had a reversible inhibitory effect on herpes simplex virus capsid transport. Herpes simplex virus can use endocytic or nonendocytic pathways to enter cells. These distinct entry routes were both dependent on proteasome-mediated proteolysis. In addition, herpes simplex virus successfully entered cells in the absence of a functional host ubiquitin-activating enzyme, suggesting that viral entry is ubiquitin independent. Herpes simplex virus immediate-early protein ICP0 is a multifunctional regulator of herpes simplex virus infection. Late in infection ICP0 interacts dynamically with cellular proteasomes. ICP0 has a RING finger domain with E3 ubiquitin ligase activity that is necessary for its IE functions. The fundamental and functional properties of ICP0 that is present in the virion tegument layer have not been well characterized. For these reasons, I sought to characterize tegument ICP0 and determine the role of tegument ICP0 during proteasome-dependent entry of herpes simplex virus. Protein compositions of wild-type and ICP0 null virions were similar, suggesting that the absence of ICP0 does not grossly impair virion assembly. Virions with mutations in the RING finger domain contained greatly reduced levels of tegument ICP0, suggesting that the domain influences the incorporation of ICP0. Virion ICP0 was resistant to removal by detergent and salt and was associated with capsids, features common to inner tegument proteins. ICP0 mutations that resulted in the absence of ICP0 in the tegument layer, allow herpes simplex virus to enter cells independently of the proteasome activity. I propose that proteasomal degradation of virion and/or host proteins is regulated by ICP0 to allow for efficient delivery of incoming herpes simplex virus capsids to the nucleus.

No description available.

Infection of most cell types with herpes simplex virus (HSV) mutants lacking the activation functions of VP16 and/or ICP0 results in repression of viral gene expression. However, the human osteosarcoma cell line U2OS supports the replication of VP16 and ICP0 mutants to nearly wild type levels. Prior to the studies presented in this thesis, the basis for the permissivity of U2OS cells to VP16 and ICP0 mutants had not been explored. Here, somatic cell fusion assays were used to determine that U2OS cells support the replication of VP16 and ICP0 mutants due to a defect in an innate gene silencing mechanism. The artificial induction of interferon stimulated genes that occurs during the somatic cell fusion assays is not the basis for the observed repression of viral gene expression. As one means of identifying components of the antiviral pathway defective in U2OS cells, restrictive cell types were treated with kinase inhibitors and infected with VP16 and/or ICP0 mutants. Although several compounds were identified which compensate for the defect in gene expression of VP16 mutants, these drugs also stimulate mutant virus gene expression in U2OS. Thus, U2OS are most likely not defective in the cellular signalling pathway(s) targeted by these compound(s). Finally, the importance of VP16 and ICP0 in modulating chromatin structure on the viral genome in both restrictive and permissive cells was examined, uncovering an essential role for both proteins in altering histone occupancy and acetylation levels. Importantly, U2OS cells have a defect in the chromatin-based pathway targeted by ICP0. However, evidence suggests that the ability of VP16 and ICP0 to affect histone occupancy and acetylation levels is not required for viral gene expression. Taken together, the results of this thesis demonstrate that U2OS cells support the replication of VP16 and ICP0 mutants due to a defect in an innate antiviral mechanism which does not involve the targets of several well characterized kinase inhibitors. The significance of the defect in a chromatin-based pathway targeted by ICP0 in U2OS cells remains to be elucidated. / Virology