The herpes simplex virus type 2 (HSV-2) UL21 protein is conserved between all members of the Alphaherpesvirinae subfamily. Although UL21 is essential for virus propagation in HSV-2, its function in viral replication is poorly understood. Cells infected with HSV-2 strains lacking UL21 exhibit an approximate two-hour delay in viral gene expression that cannot be explained by a defect in virus entry or capsid engagement with, or movement along, microtubules. However, we noted a defect in the ability of UL21 knockout (KO21) capsids to associate with the nucleus after infection. We found that the delay in viral gene expression was not directly due to the absence of UL21 insofar as cells stably expressing UL21 could not complement the delay in gene expression. We suggest that the KO21 delay in gene expression is due to alterations in virion composition and that in the absence of UL21, a key virion component required for the timely delivery of capsids to the nucleus fails to be packaged into virions. Interestingly, at late times after infection, levels of viral proteins in KO21 infected cells reach wild-type levels, indicating that a secondary function is responsible for the essential nature of UL21. We found that at late times post-infection KO21 infected cells accumulated capsids in the nucleus but these fail to reach the cytoplasm and mature into infectious virions. Thus, we hypothesize that the essential function of UL21 is to facilitate capsid trafficking from the nucleus to the cytoplasm. Moreover, the viral glycoproteins gD and gC were retained in the endoplasmic reticulum, and were under-glycosylated in KO21 infected cells. It is therefore possible that the absence of UL21 prevents the targeting of glycoproteins to the inner nuclear membrane, preventing the formation or function of the nuclear egress complex. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2013-09-10 16:20:32.162

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)

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)

Background: Herpes simplex virus type 2 (HSV-2), the primary cause of genital herpes, is one of the most widespread, lifelong sexually transmitted infections (STIs). Incidence is disproportionately higher in women compared to men, so a better understanding of vaginal transmission, the primary mode for HSV-2 infection in women, is crucial for developing preventative strategies. Female sex hormone, estrogen (E2), has been shown to play a protective role against sexually transmitted viral infections and previous studies have shown that vaginal epithelial cells treated with E2 are protected against HSV-2 infection; however, the underlying mechanism of E2 protection remains unclear, so a transcriptome analysis followed by functional studies was performed. Method of Study: In this study, VK2/E6E7 (vaginal epithelial) cells were used to study HSV-2 entry, infection and replication. VK2s were grown in Air-Liquid-Interface (ALI) cultures, allowing for their proliferation and stratified layer formation in transwells; closely mimicking physiological conditions. Media was supplemented with no hormone (NH) or physiological concentrations of E2, P4 and MPA for 7 days. After 24 hours of HSV-2 infection in these cultures, VK2 cells were lysed and processed for RNA isolation. We performed a comprehensive genome-wide microarray to profile gene expression of VK2 cells pre-treated with and without E2, prior to, and following HSV-2 infection. For data analysis, “R” software was used to perform all pre-processing steps and normalization. Gene Set Enrichment Analysis (GSEA) was performed to identify potential cellular pathways regulated by E2 after infection using the Hallmark database, relative to NH conditions. Immunofluorescence staining was used for functional analysis to confirm transcriptomic data. After selecting a pathway for investigation, small-molecule inhibitors and activators of this pathway were used in combination with NH or E2. Vero plaque assay and HSV-2-GFP infection were used to identify examine the protective effects of E2 and the selected pathway. In addition, we also used siRNA to specifically knockdown proteins part of the pathway and investigate the specific effects on protection against HSV-2. Results: Microarray analysis indicated that exposure to HSV-2 in the presence of E2 resulted in differential transcriptional profile compared to NH and P4. GSEA assigned one of the highest enrichment scores to the p53 pathway compared to other pathways under the influence of E2 compared to NH, following HSV-2 infection. Studies to correlate bioinformatic results with functional analysis showed significant increase in p53 protein expression after E2 treatment compared to NH. Vero plaque assay demonstrated 10-fold decrease in viral replication following E2 treatment as well as by direct activation of p53 in absence of E2. In contrast, p53 inhibition even in the presence of E2 resulted in 100-fold increased viral replication compared to E2 alone, suggesting that the p53 is involved in E2-mediated protection. We deduced that E2 particularly affects HSV-2 replication and not entry into VK2 cells. We also found that BST2 is strongly regulated by E2-mediated p53 and also contributes to protection against HSV-2. Lastly, we demonstrated that E2 demonstrates anti-inflammatory effects that correlate with its increase in barrier integrity seen with VK2s. Conclusions: With bioinformatic and functional analysis, we found that E2 provides protection through the p53 pathway, as well as through downstream BST2. Our data provides the first comprehensive overview of host cellular responses to HSV-2 and female sex hormones at a transcriptional level and highlights the protective role of E2-mediated p53 pathway. This study is the first to deduce the antiviral mechanism of E2 against HSV-2 infection in human vaginal epithelial cells. / Thesis / Master of Science (MSc)

M.P.H., Faculty of Health Sciences, University of the Witwatersrand, 2009 / Background: Herpes Simplex Virus type 2 (HSV-2) is a common cause of genital ulcers worldwide and has emerged as a co-factor in human immunodeficiency virus (HIV) acquisition and transmission. A study was conducted to determine the prevalence of HSV-2, its correlates, the accuracy of reported history of genital ulcer disease (GUD) to predict HSV-2 infection and the extent of symptom recognition in a clinic population in Johannesburg. Methods: 210 women aged 18 years or older were interviewed and socio-demographic, sexual behaviour and clinical information collected. Serological testing for HSV-2 and HIV infections was performed, but only where sera were available for the latter. Factors associations with HSV-2 infection were assessed using logistic regression to estimate odds ratios (OR) and 95% confidence intervals (CI). The sensitivity, specificity, predictive values and likelihood ratios of a history of GUD were calculated. Results: The estimated sero prevalence of HSV-2 was 73% (95% CI 67% - 79%). Few participants, 13/206 (6%) participants had knowledge of genital herpes. Only 9/203 (4%) participants recognised lesions of genital herpes following education and counselling about HSV-2 infection. HSV-2 infection was associated with older age(>25 years of age) OR 2.6 (95% CI 1.4-5.0), spending more than 2 nights away from home, OR 6.0 (95% CI 1.0-62.7), having more than 2 sexual lifetime partners, OR 2.2 (95% CI 1.1-3.9), a history of an STI in the past 3 months ,OR 3.6 (95% CI 1.2-9.5) and HIV infection, OR3.3( 95%CI 1.4-7.9). A history of genital ulceration performed poorly as a predictor of HSV-2 seropositivity; the sensitivity was 7% and specificity was 96%. Conclusion: HSV-2 prevalence was high and few participants were aware of their infection. HVS-2 infection was associated with risky sexual behaviour .A history of genital ulcer disease was not sufficient as a diagnostic tool for HSV-2 infection. Public health interventions should focus on behavioural modification and increasing awareness of genital herpes. HSV-2 management should be incorporated into HIV care and STI protocols.

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No. of bitstreams: 1 Dissertação - Tatiana Amaral Pires de Almeida.pdf: 3233406 bytes, checksum: e04eb0576f711f1ed5bbe73ae78b4932 (MD5) Previous issue date: 2010-08-20 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / The Herpes Simplex Virus type 2 (HSV-2) is a widespread, incurable agent that establishes latency in nervous cells and is responsible for the majority of genital herpes cases. The UL23 is a polymorphic gene, which encodes the viral thymidine kinase (TK) of the Herpes virus. TK has six conserved regions, including two active sites, which plays an important role in acyclovir (ACV) metabolism. Therefore, some mutations in UL23 are implicated with ACV resistance phenoty pe. In this work, we analyzed 67 samples collected between Ap ril 2008 and September 2009 at the STI/STD clinic of “Alfredo da Matta” Foundation, in Manaus, Amazonas, Brazil obtained from HSV-2 genital ulcer patients. In order to characterize polymorphisms, all sp ecimens were processed for total nucleic acid isolation, submitted to PCR amplification targeting the complete ORF of UL23 gene followed by automated nucleotide sequencing with dideoxy chain terminators. Sequences analysis shown that 45 sequences were 100% similar to the reference strain “HG52”. The remaining 22 sequences (32.8%) showed at least one nucleotide substitution; none insertions or deletions were found. Amino acids substitutions were detected in 19 out of 22. The mutation Gly39Glu was the most frequently detected (16/19), followed by Asn78Asp (5/19); Gly39Glu and Asn78Asp were simultaneously found in four samples. Deduced amino acids sequences were aligned with 66 available on GenBank from African and American continents. A total of 23 variable sites were identified and, once more, Gly39Glu mutation was the most frequent found (74/133). Substitutions previously associated with ACV resistance were not detected, however, Gly39Glu was recently described as a probable resistant mutation. Another four mutations observed: His4Tyr, Met70Arg, Asn245Ser and Ala366Val had not been previously described. The theoretical models of TK’s structure built for seven samples showed that amino acids substitutions were found away from the conserved and/or catalytic sites of TK, with the exception of Arg338Gln which was nearly from ATP-binding site. Mutations associated with ACV resistance were not detected. However one of the mutations found, Gly39Glu, was p reviously associated with this resistance phenoty pe. Another four mutations found in his work: His4Tyr, Met70Arg, Asn245Ser and Ala366Val had not been previously described elsewere. To our knowledge, this is the first study on UL23 polymorphisms on Brazilian HSV-2 samples, a research field considered as a priority by WHO HIV/STI program. / latência em células nervosas sendo responsável pela maioria dos casos de herpes genital. O UL23 é um gene polimórfico que codifica a timidinaquinase viral (TK) do Herpesvírus. A TK possui seis regiões conservadas, incluindo dois sítios ativos; esta enzima desempenha um papel importante no metabolismo do aciclovir (ACV). Consequentemente, algumas mutações no gene UL23 estão relacionadas a fenótipos de resistência ao ACV. Nesta dissertação, analisamos 67 amostras coletadas entre Abril de 2008 e Setembro de 2009 na clínica de DST/IST da Fundação “Alfredo da Matta”, em Manaus, Amazonas, Brasil, obtidas de pacientes com úlceras genitais por HSV-2. Com o objetivo de se caracterizar polimorfismos, todos os espécimes foram processados para extração total de ácido nucleico e submetidos à amplificação da CDS do gene UL23 pela técnica de PCR, seguida de sequenciamento nucleotídico automatizado utilizando-se terminadores dideóxi. As análises de sequências mostraram que 45 delas foram 100% similares à cepa-referência “HG52”. As 22 sequências restantes (32,8%) mostraram pelo menos uma substituição de nucleotídeo; nenhuma inserção ou deleção foi encontrada. Substituições de aminoácidos foram detectadas em 19 dessas 22 sequências. A mutação Gly39Glu foi a mais frequente (16/19), seguida pela Asn78Asp (5/19); Gly39Glu e Asn78Asp foram encontradas simultaneamente em quatro amostras. Sequências deduzidas de aminoácidos foram alinhadas com 66 sequências Africanas e Americanas disponíveis no GenBank. Um total de 23 sítios variáveis foram identificados e, mais uma vez, a mutação Gly39Glu foi a mais frequente (74/133). Substituições previamente associadas à resistência ao ACV não foram detectadas, entretanto, a Gly39Glu foi recentemente descrita como uma provável mutação de resistência. Outras quatro mutações observadas, His4Tyr, Met70Arg, Asn245Ser e Ala366Val, foram descritas pela primeira vez por este trabalho. Nos modelos teóricos da estrutura da TK, construídos para sete amostras, foi possível visualizar que as substituições de aminoácidos estavam situadas longe dos sítios ativos/conservados desta enzima, com exceção da mutação Arg338Gln que apresentou maior proximidade com o sítio de ligação de ATP. Não foram detectadas mutações sugestivas de resistência ao ACV. Ao nosso conhecimento, este é o primeiro estudo de polimorfismos do gene UL23 do HSV-2 em amostras brasileiras, um campo considerado prioridade pelo Programa de HIV/IST da OM S.

Encephalitis caused by the alphaherpes viruses HSV 1, HSV 2 and VZV can be devastating and rapid, accurate diagnosis is required. Whilst existing molecular techniques are invaluable in diagnosing acute disease, detection of antibody is needed to confirm infection and to make a diagnosis after the acute stage or during post-infectious encephalitis. Current immunoassays are limited by the volume of sample required. The aim of this project was to develop a rapid, accurate, low sample volume assay to improve diagnosis using Luminex technology.The immunodominant proteins of HSV and VZV, glycoprotein D (gD) and glycoprotein E (gE), were expressed in insect cells using a baculovirus expression vector. Expressed proteins were purified, characterised and used to develop in-house enzyme-linked immunosorbent assays (ELISA) to detect HSV and VZV type-specific antibodies. The performance of each newly developed in-house ELISA was compared with commercial ELISA assays using well characterised serum panels. An excellent correlation between the in-house ELISAs and the commercial ELISA assays (100% for HSV gD and 99% for VZV gE) was observed. To differentiate between HSV-1 and HSV-2 a new commercial ELISA assay (Omega) utilising a branched chain peptide (peptide 55 which provides immune selection of HSV-2 specific antibody) was evaluated against two commercially available HSV-2 ELISA assays. The Omega assay showed an overall agreement of 97.6% with Western blot and other ELISA assays. The two expressed proteins, together with peptide 55, were used to develop a triplex fluorescent microbead immunoassay for the simultaneous detection and quantitation of anti-viral antibody in human sera. Initially a monoplex assay for each analyte was developed and optimised individually and then the three assays were mixed together in a triplex assay. Results for HSV-1 gD and VZV gE obtained from the triplex assay showed a 100% agreement with HSV-1 and VZV in-house ELISA results. In the case of peptide 55, the triplex assay results showed better sensitivity than the Omega ELISA assay with an overall agreement with Western blot and other assays of 98.4%. In addition, in order to facilitate the diagnosis of alphaherpesviruses CNS infections the triplex assay was joined together with a biplex fluorescent microbead immunoassay designed for detecting and measuring human IgG and albumin in CSF and serum samples. The sensitivity and reproducibility of the resultant five-analyte multiplex immunoassay and the previous triplex assays were compared and found to have equivalent sensitivity and specificity. The sensitivity and minimal sample requirements of the new assay suggests that it will be a powerful tool for the diagnosis and study of both acute and post-infectious viral encephalitis.

Background: HSV-2 was estimated to infect 491 million people worldwide, with women disproportionately affected by HSV-2. Understanding factors that influence susceptibility to HSV-2 in women is important in preventing infections. Through various studies, the progestin-based contraceptive DMPA exhibited immunosuppressive effects, and has shown increased susceptibility to HIV and HSV-2. Studies comparing DMPA to other contraceptives like NET suggest that NET may be safer. In vivo NET effects have not been characterized thoroughly to better understand the effect of NET on susceptibility to HSV-2. Therefore, this study aimed to compare the effects of NET and DMPA in mouse models that affect susceptibility to HSV-2. We hypothesized that NET treated mice will have decreased susceptibility to HSV-2 compared to DMPA but elevated compared to normal mice. Method of study: Ovariectomized mice were treated with DMPA (2mg) and NET (2 mg injections, 2.5 mg pellets or 5 mg pellets) for 10 days and intravaginally immunized with HSV-2 TK-, then intravaginally challenged with WT HSV-2 ~4-7 weeks later. Primary intravaginal WT HSV-2 challenges were conducted in ovariectomized and normal mice after 10 days of DMPA and NET treatment. Viral titers, pathology and survival were examined. Mucus production in the vagina was investigated through immunohistology. Effects of hormones on immune cells were explored in the lymph nodes, spleens, and vaginal tracts through flow cytometry. Results: Increased mucus was consistently observed in the vaginal tracts of mice after treatment with NET 2.5 mg and 5 mg treated mice, but not with DMPA Therefore, NET treated mice displayed reduced viral shedding and delayed pathology compared to DMPA treated mice. No significant changes occurred in immune cells analyzed post DMPA and NET treatment, although there were trends of increased T cells in progestin treated mice. However, more experiments need to be conducted to confirm observed trends. Conclusion: NET treatment in mice results in mucus production in the vaginal tract, a potential mechanism impeding intravaginal HSV-2 infection and could be applied to other STIs. This provides insight into protective effects of NET compared to DMPA allowing women to make informed decisions regarding hormonal contraceptives. / Thesis / Master of Science in Medical Sciences (MSMS)

No description available.

Herpes Simplex virus (HSV) virions contain the protein vhs (virion host shutoff), which is known to trigger rapid shutoff of host protein synthesis and accelerated decay of viral and cellular mRNAs. HSV-1 strains generally cause weaker shutoff than HSV-2 strains. HSV viruses lacking the VP16 viral transactivator gene show uncontrolled shutoff late in infection (Lam et al., 1996); vhs is known to bind to VP16 (Smibert et al., 1994). In vitro experiments demonstrated that HSV-1 vhs protein (vhsTl) and an intertypic HSV-2 G vhs protein (vhsT2) did not differ in ability to effect mRNA degradation in a rabbit reticulocyte lysate (RRL) assay system, suggesting that virion factors might influence vhs shutoff phenotype. To investigate the possibility that VP 16 influences early shutoff during HSV infection, a virus was constructed containing the HSV-2 strain G VP16 in place of HSV-1 VP16. This virus (8MA2R) grew in a noncomplementing cell line and was unaltered in shutoff phenotype compared to wt strain. Cotransfection assays demonstrated vhsT2 had greater shutoff ability than vhsT1, and this was confirmed in a viral system by constructing intertypic viruses containing portions of the HSV-2 G vhs ORF in a vhs-null HSV-1 tk locus. All intertypic constructs conferred increased shutoff ability relative to vhsT 1, indicating that the strong shutoff ability of HSV -2 vhs is distributed along much of the vhs ORF. Also, to confirm the observation that UL13 null viruses show a lack of shutoff which is not due to lack of vhs in the virion (Overton et al., 1994) a number of wildtype viruses and their UL13-null derivatives were tested for ability to shutoff host translation. All showed near-wt or wt levels of shutoff. / Thesis / Master of Science (MS)