Background: Over the last twenty years the incidence of herpes zoster (HZ) infection, also known as shingles, has been increasing among adults for unknown reasons. The economic burden of HZ is currently estimated at over $1 billion per year in the United States (U.S.) and is expected to increase as the susceptible adult population ages. HZ is caused by a re-activation of the varicella zoster virus (VZV), chicken pox, and more than 95% of adults living today carry the virus with a lifetime risk of 1 in 3 for developing HZ. In 2006 the FDA approved a vaccine for the prevention of HZ in adults 60 years and older and in 2011 approval was expanded to include adults age 50-59 years. Since 2006 rates of adult immunization for HZ have been modest, as of 2015 approximately two-thirds of the US population ≥ 60 are still unvaccinated and more than 94% of those ages 50-59 have not been vaccinated. There is now accumulating evidence of a significantly elevated risk of ischemic stroke (IS) within the first 12 months following infection with HZ. Every 40 seconds someone in the U.S. suffers a stroke with an estimated 795,000 strokes per year. In the U.S. stroke is a significant cause of disability with costs estimated at $33 billion per year including cost of healthcare, medication, and lost productivity. As the population in the U.S ages, the risk of both HZ infection and stroke will increase significantly thus impacting mortality, morbidity, and healthcare costs. The CDC Advisory Committee for Immunization Practices (ACIP) currently recommends routine vaccination against HZ for adults ≥ 60 but does not recommend vaccination for adults age 50-59 years and does not provided any guidance or recommendations for adults who may be at increased risk of stroke associated with HZ infection. The current ACIP vaccination recommendations for HZ are predominately based on clinical trial efficacy data and cost-effectiveness analyses (CEAs) in adults ≥ 60. These prior analyses did not included costs associated with the recent evidence demonstrating increased risk of stroke up to one year following HZ infection. Aims: The objectives of this study were as follows; 1) To assess the cost-effectiveness of a targeted HZ vaccination strategy for adults age 50-59 years at increased cardiovascular (CV) risk in whom vaccination is approved but not recommended; 2) To develop a white paper directed at payers, providers, and policy makers translating the findings from the analysis into appropriate population health dissemination, implementation, and adoption priority recommendations. Methods: A decision analytic Markov Model (MM) was used to compare costs and outcomes between two vaccination strategies; usual-care (no current vaccine recommendation) and targeted vaccination in adults age 50-59 years with cardiovascular disease (CVD) in a hypothetical cohort of 100,000 adults age 50-59 years. The private payer perspective was used as it best represents this population of adults age 50-59 years who are predominately employed and covered under employer sponsored commercial insurance. The simulated cohort was assessed for incidence of IS within 12 months following HZ infection occurring within the fifth decade of life. Risk was assessed from the age at entry to the analysis, median age 55, up to age 60 using TreeAge Pro 2017 software. The cohort was then aged out to 100 years or death, whichever came first. Costs were calculated using 2016 U.S. dollars. Findings: As it relates to aim one, compared to usual-care targeting HZ vaccination in adults age 50-59 years with prevalent CVD was cost-effective with an incremental cost-effectiveness ratio (ICER) of $55,517 per quality of life-year (QALY) gained which falls well below the standard willingness-to-pay (WTP) threshold of $100,000 utilized in previous HZ CEAs (Le & Rothberg, 2015, 2016; Pellissier, Brisson, & Levin, 2007). The incremental cost of vaccinating the target population using a benchmark vaccination rate of 60% was $30.59 per person compared to $12.98 in the usual-care group with ICERs of $55,517 and $55,470 respectively. Moreover, when comparing the cost of universal vaccination in the entire 50-59 year old cohort cost-effectiveness was maintained with an incremental cost of $176.51 per person and an ICER of $55,523. Adopting the targeted strategy resulted in 162 fewer cases of HZ and 14 fewer strokes per 100,000 persons. Regarding aim two, following safety and efficacy, cost-effectiveness analysis are considered an essential metric in vaccine policy making and a substantial driver of vaccine adoption by policymakers, payers, and providers. Translating these favorable cost-effectiveness findings to policymakers, payers, and providers is necessary to help close the adoption curve gap in order to facilitate and inform effective and timely implementation strategies for HZ vaccination in this targeted population. Conclusions: This study demonstrated that targeted HZ vaccination in patients age 50-59 years at increased CV risk is cost-effective and thus updating ACIP policy recommendations regarding vaccination in this population for whom the vaccine is currently FDA approved but not recommended should be considered. Furthermore, this study showed that universal vaccination in the general 50-59 year old population is cost-effective. Given the very limited data on cost-effectiveness of HZ vaccination in adults age 50-59 years, which has resulted in a lack of recommendation for this population, and recent evidence of IS risk the results of this study demonstrating cost-effectiveness of a targeted HZ vaccination strategy directly support the National Adult Immunization Plan (NAIP) to improve adult immunization uptake by providing economic evaluations which can be used to inform policymakers, payers, and providers.

Herpes simplex virus type 1 (HSV-1) is a common human pathogen that establishes life-long latent infection in sensory neurones. This makes it potentially useful as a gene therapy vector to target neuronal cells. HSV-1 enters cells by membrane fusion, the viral envelope and most tegument proteins dissociate, and the capsid is transported to the cell nucleus to establish infection. There is increasing evidence that the retrograde transport of HSV-1 along sensory axons is mediated by cytoplasmic dynein, but the viral and cellular proteins involved are not known. Cytoplasmic dynein is the major molecular motor involved in minus-end-directed cellular transport along microtubules. It is a large complex molecule, with heavy chains providing motility, while intermediate and light chains are involved in specific cargo binding. A library of HSV-1 capsid and tegument structural genes was constructed and tested for interaction with dynein subunits in a yeast two-hybrid system. A strong interaction was demonstrated between the HSV-1 outer capsid protein VP26 (UL35), as well as the tegument protein VP11/12 (UL46), with the homologous 14 kDa dynein light chains rp3 and Tctex1. In vitro pull-down assays confirmed binding of VP26 to rp3, Tctex1 and cytoplasmic dynein complexes. Recombinant HSV-1 capsids +/- VP26 were used in similar pull-down assays. Only VP26+ capsids bound to rp3. Recombinant HSV-1 capsids were microinjected into living cells and incubated at 37�C. After 1 h capsids were observed to co-localise with rp3, Tctex1 and microtubules. After 2 or 4 h VP26+ capsids had moved closer to the cell nucleus, while VP26- capsids remained in a random distribution. Our results suggest that the HSV-1 outer capsid protein VP26 mediates binding of incoming capsids to the retrograde motor cytoplasmic dynein during cellular infection, through interactions with dynein light chains. It is hoped that these findings will help in the development of a synthetic viral vector, which may allow targeted gene therapy in patients with neurological diseases.

Bibliography: leaves 92-118. / xii, 118, [20] leaves, [12] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Reports a method for the study of HSV-1 transcripts during latency. High resolution non-isotopic in situ hybridization (ISH) is used to study the intracellular location of HSV-1 latency associated transcripts (LATs) in primary sensory neurons of latently infected mice and humans. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1995?

<p>Viruses depend on their hosts for the production and spread of new virus particles. For efficient virus replication, the viral genes have adapted the strategy of being recognized and processed by the cellular biosynthetic machineries. Viruses therefore provide an important tool to study the cellular machinery regulating gene expression. In this thesis, we have used two model DNA viruses; herpes simplex virus (HSV) and adenovirus, to study RNA processing at the level of pre-mRNA splicing in mammalian cells. </p><p>During a lytic infection, HSV cause an almost complete shut-off of host cell gene expression. Importantly, HSV infection cause inhibition of pre-mRNA splicing which is possibly advantageous to the virus, as only four HSV genes contain introns. </p><p>The HSV immediate early protein, ICP27, has been shown to modulate several post-transcriptional processes such as polyadenylation and pre-mRNA splicing. We have studied the role of ICP27 as an inhibitor of pre-mRNA splicing.</p><p>We show that ICP27 inhibits pre-mRNA splicing <i>in vitro</i> in the absence of other HSV proteins. We further show that ICP27 inhibits splicing at the level of spliceosome assembly. Importantly, ICP27 induced inhibition of splicing can be reversed, either by the addition of purified SR proteins or by the addition of an SR protein specific kinase, SRPK1. We propose that SR proteins are prime candidates as mediators of the inhibitory effect of ICP27 on pre-mRNA splicing. </p><p>In order to learn more about how splicing is organized in the cell nucleus <i>in vivo</i>, we investigated how cellular splicing factors are recruited to sites of transcription and splicing in adenovirus infected cells using confocal microscopy. Our results showed that the SR proteins, ASF/SF2 and SC35, are efficiently recruited to sites in the nucleus where adenovirus genes are transcribed and the resulting pre-mRNAs are processed. Our results demonstrate that only one of the two RNA recognition motifs (RRMs) present in the ASF/SF2 protein is required for its recruitment to active sites of splicing. The arginine/serine rich (RS) domain in ASF/SF2 is redundant and insufficient for the translocation of the protein to active viral polymerase II genes in adenovirus infected cells.</p>

Transcription and DNA replication of herpes simplex virus type 1 (HSV-1) occur in nuclear domains adjacent to structures named ND10. The HSV-1 single-stranded DNA binding protein ICP8 localizes to these nuclear domains to direct the assembly of the pre- and replication compartments. Inhibition of cyclin dependent kinases with roscovitine inhibits HSV-1 DNA replication, even in the presence of all required HSV-1 proteins, at an unidentified step. Here I show that roscovitine inhibits the localization of pre-expressed ICP8 to new replication sites. Therefore, the inhibition of HSV-1 DNA replication occurs at a step prior to initiation. I next evaluated the mechanisms of inhibition of proper ICP8 localization. ICP8 was extracted at lower salt concentrations from roscovitine-treated than untreated cells, but the affinity of ICP8 for ssDNA in vitro was not affected. I propose that roscovitine inhibits HSV-1 DNA replication by inhibiting DNA accessibility. I also discuss alternative mechanisms.

Viruses depend on their hosts for the production and spread of new virus particles. For efficient virus replication, the viral genes have adapted the strategy of being recognized and processed by the cellular biosynthetic machineries. Viruses therefore provide an important tool to study the cellular machinery regulating gene expression. In this thesis, we have used two model DNA viruses; herpes simplex virus (HSV) and adenovirus, to study RNA processing at the level of pre-mRNA splicing in mammalian cells. During a lytic infection, HSV cause an almost complete shut-off of host cell gene expression. Importantly, HSV infection cause inhibition of pre-mRNA splicing which is possibly advantageous to the virus, as only four HSV genes contain introns. The HSV immediate early protein, ICP27, has been shown to modulate several post-transcriptional processes such as polyadenylation and pre-mRNA splicing. We have studied the role of ICP27 as an inhibitor of pre-mRNA splicing. We show that ICP27 inhibits pre-mRNA splicing in vitro in the absence of other HSV proteins. We further show that ICP27 inhibits splicing at the level of spliceosome assembly. Importantly, ICP27 induced inhibition of splicing can be reversed, either by the addition of purified SR proteins or by the addition of an SR protein specific kinase, SRPK1. We propose that SR proteins are prime candidates as mediators of the inhibitory effect of ICP27 on pre-mRNA splicing. In order to learn more about how splicing is organized in the cell nucleus in vivo, we investigated how cellular splicing factors are recruited to sites of transcription and splicing in adenovirus infected cells using confocal microscopy. Our results showed that the SR proteins, ASF/SF2 and SC35, are efficiently recruited to sites in the nucleus where adenovirus genes are transcribed and the resulting pre-mRNAs are processed. Our results demonstrate that only one of the two RNA recognition motifs (RRMs) present in the ASF/SF2 protein is required for its recruitment to active sites of splicing. The arginine/serine rich (RS) domain in ASF/SF2 is redundant and insufficient for the translocation of the protein to active viral polymerase II genes in adenovirus infected cells.

L'encéphalite herpétique (EH), causée par l'herpès simplex virus-1 (HSV-1), peut résulter de défauts monogéniques de l'immunité médiée par TLR3. L'induction d'interférons (IFNs)-α/β ou -λ via TLR3 est cruciale à la protection après infection primaire avec HSV-1 dans le système nerveux central (SNC). Nous décrivons deux enfants avec l'EH portant différentes mutations hétérozygotes (D50A et G159A) dans TBK1, encodant TANK-Binding Kinase 1, une kinase aux carrefours de multiples voies de signalisation induisant des IFNs. Les deux allèles mutants de TBK1 sont perte-de-fonction par des mécanismes différents: instabilité de la protéine (D50A) ou perte d'activité kinase (G159A). Ces allèles sont associés à un trait autosomal dominant (AD) par des mécanismes différents: haplotype-insuffisance (D50A) ou dominance négative (G159A). Un défaut de réponses à poly(I:C) par TLR3 est observable dans les fibroblastes hétérozygotes pour G159A, et non pour D50A TBK1. Néanmoins, la réplication virale et la mortalité cellulaire après infection par deux virus dépendants de TLR3 (HSV-1 et VSV) étaient élevées dans les fibroblastes des deux patients. Ces phénotypes peuvent être sauvés par IFN-α2b. De plus, la production d'IFNs en réponse à des agonistes et virus indépendants de TLR3 est maintenue dans les PBMCs et fibroblastes des patients. Le phénotype cellulaire restreint, partiel représente ainsi le phénotype clinique de ces patients, limité à l'EH. Ces données identifient la déficience partielle AD de TBK1 comme une nouvelle étiologie génétique de l'EH de l'enfance, et indiquent que TBK1 est essentiel pour le contrôle de HSV-1 dans le SNC, médié par TLR3 et dépendant des IFNs

Fracture repair is a regenerative event that involves the precise coordination of a variety of cells for successful healing process. Within the microstructure hierarchy of bone repair, the predominant cells involved include the chondrocytes, osteocytes, osteoblasts, and osteoclasts. Although the role of osteoblasts during fracture healing has been previously shown, their role during the initiation phase of endochondral fracture repair remains unclear. In order to study the role of osteoblasts during fracture repair, we used a transgenic mouse model expressing the herpes simplex virus thymidine kinase gene in early differentiating osteoblasts, which allows conditional ablation of cells in osteoblastic lineage upon treatment with the Gancicolvir drug. Results from this study suggest that not only are osteoblasts required in later stages of fracture repair as the medium for bone synthesis, and osteoclast activation during bone remodelling, but could also be required for the initiation and advancement of the endochondral ossification process.

Fracture repair is a regenerative event that involves the precise coordination of a variety of cells for successful healing process. Within the microstructure hierarchy of bone repair, the predominant cells involved include the chondrocytes, osteocytes, osteoblasts, and osteoclasts. Although the role of osteoblasts during fracture healing has been previously shown, their role during the initiation phase of endochondral fracture repair remains unclear. In order to study the role of osteoblasts during fracture repair, we used a transgenic mouse model expressing the herpes simplex virus thymidine kinase gene in early differentiating osteoblasts, which allows conditional ablation of cells in osteoblastic lineage upon treatment with the Gancicolvir drug. Results from this study suggest that not only are osteoblasts required in later stages of fracture repair as the medium for bone synthesis, and osteoclast activation during bone remodelling, but could also be required for the initiation and advancement of the endochondral ossification process.

B virus diagnosis presents a challenge largely complicated by the asymptomatic infection of rhesus macaques, and extremely pathogenic fatal infections in humans. Humoral detection of antibodies is generally performed using whole virus antigen for which preparation requires strict biosafety measures and specialized BSL-4 facilities. As an alternative to utilizing B virus antigen, we describe the production of a truncated form of B virus envelope glycoprotein D, gD 287, in a baculovirus expression system, and evaluate its diagnostic potential as an antigen in recombinant ELISA. After purification and characterization, gD 287 was tested using 22 negative and 72 positive macaque sera samples previously classified using the traditional method. We find that sensitivity and specificity of the recombinant ELISA are dependent on antibody titer of tested serum and gD 287 shows good to excellent predictive potential for identification of positive sera with titers higher than 500.