Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Mar 10

Thèse ou mémoire avec insertion d'articles / Le virus respiratoire syncytial (VRS) et le métapneumovirus humain (MPVH) sont deux pathogènes respiratoires majeurs qui représentent un fardeau important pour la santé publique dans le monde entier, affectant particulièrement les populations vulnérables telles que les nourrissons, les personnes âgées et les patients immunodéprimés. Les deux virus appartiennent à la famille des *Pneumoviridae* (pneumovirus) et sont relativement similaires dans leur structure et leur réplication. Malgré plus de 60 ans de recherche dans ce domaine, les deux premiers vaccins contemporains pour la prévention du VRS ont été approuvés cette année, mais il n'existe toujours pas de vaccin contre le MPVH. Cette thèse donne un aperçu complet de la virologie moléculaire du VRS et du MPVH, de l'épidémiologie, du fardeau de la maladie, des modèles d'infection, de la réponse immunitaire et du paysage actuel des modalités de traitement et de prophylaxie du VRS et du MPVH, en mettant l'accent sur le développement d'un vaccin. Notre équipe de recherche a déjà développé Metavac®, un candidat vaccin vivant atténué contre le MPVH qui s'est avéré sécuritaire, immunogène et protecteur contre l'infection par le MPVH chez la souris. Cette thèse présente une tentative réussie de conception, de génération et de caractérisation d'un vaccin chimérique atténué MPVH/VRS basé sur la plateforme d'expression Metavac® en vue de son utilisation comme vaccin chimérique atténué bivalent contre les deux pneumovirus. Le projet de recherche comprend trois parties principales : I) Biologie moléculaire : L'étude de la génétique des pneumovirus et la tentative de trouver une localisation appropriée dans le génome du MPVH pour l'insertion de l'antigène principal du VRS. II) *II) In vitro :* Génération d'un virus viable par génétique inverse et production d'un stock de virus concentré pour son utilisation comme vaccin chimérique atténué MPVH/VRS. Caractérisation des virus chimériques. III) *In vivo*: Vaccination de souris BALB/c avec des virus chimériques MPVH/VRS et évaluation de leur efficacité protectrice contre le défi létal MPVH et VRS. / Respiratory Syncytial Virus (RSV) and Human Metapneumovirus (HMPV) are two major respiratory pathogens that pose a significant public health burden worldwide, particularly affecting vulnerable populations such as infants, elderly individuals, and immunocompromised patients. Both viruses belong to the family *Pneumoviridae* (pneumovieus) and are relatively similar in structure and replication. Despite over 60 years of research in the field, the first two contemporary vaccines for RSV prevention have been approved this year, and yet there is still no vaccine available against HMPV. This thesis provides a comprehensive overview of RSV and HMPV molecular virology, epidemiology, disease burden, models of infection, immune response, and the current landscape of RSV and HMPV treatment and prophylactic modalities with a focus on vaccine development. Our research team has previously developed Metavac®, a live attenuated HMPV vaccine candidate that proved safe, immunogenic, and protective against HMPV infection in mice. This work presents a successful attempt to design, generate, and characterize a chimeric attenuated HMPV/RSV virus based on the Metavac® expression platform for its use as a bivalent attenuated chimeric vaccine against both pneumoviruses. The research project includes three main parts: IV) Molecular biology: The study of pneumovirus genetics and the attempt to find an appropriate localization in the genome of HMPV for the insertion of the main antigen of RSV. V) *In vitro*: A rescue of a viable virus by reverse genetics and the production of a concentrated virus stock for its use as an attenuated chimeric HMPV/RSV vaccine. Characterization of chimeric viruses. VI) *In vivo*: Vaccination of BALB/c mice with chimeric HMPV/RSV viruses and evaluation of their protective efficacy against HMPV and RSV lethal challenge.

The outer surface of the human placenta, the syncytiotrophoblast, results from the fusion of many cytotrophoblast cells such that many nuclei are contained in this layer. It is possible for these nuclei to cluster forming syncytial nuclear aggregates (SNAs). SNAs have been linked to pathology with increased numbers and earlier formation of SNAs in preeclampsia and fetal growth restriction (FGR). SNAs can be grouped into subtypes including bridges, knots and sprouts, dependent on morphology and attachment to surrounding placental villi. Little is known about SNA formation, but the pyknotic appearance of nuclei within SNAs has led to development of a hypothesis that SNAs are the terminal point of nuclear turnover in the syncytiotrophoblast. Some cytoskeletal proteins have been associated with SNAs indicating their potential involvement in SNA formation. This project aimed to uncover differences between SNA subtypes, whether the degenerate nuclear morphology represents apoptosis and to understand which mechanisms drive nuclear collection into SNAs. Experimental approaches included a review of an electron micrograph archive and application of immunohistochemical techniques to ex vivo placental tissue. A long-term explant model was developed to examine SNA development in vitro; these experiments were further explored using an isolated primary cytotrophoblast model. Nuclei within SNAs were more frequently pyknotic and less frequently eukaryotic than nuclei dispersed in the syncytiotrophoblast. However, few SNAs were positive for the cytokeratin-M30 neoepitope, a caspase dependent breakdown product of cytokeratin-18 and no subtype of SNA showed greater M30 staining than general areas of syncytiotrophoblast. There were increased syncytial knots and decreased syncytial bridges in placentas from women with preeclampsia compared to controls and FGR. While cytoskeletal proteins are seen surrounding SNAs, inhibition of actin and tubulin had no effect on SNA turnover or stability. Very limited nuclear movement was recorded from in vitro culture indicating that syncytiotrophoblast nuclei move far less than had been expected. These data suggest that cell death was not prominent within SNAs but different prevalence of subtypes were present in preeclampsia indicating that SNAs might represent larger changes in placenta structure. As nuclei moved less and SNAs were more static than expected it is suggests that SNAs are more stable than previously thought. Overall, the hypothesis that SNAs are highly active in preeclampsia is questioned and new hypotheses of the role of SNAs are considered in the light of these experimental findings, including whether they form by chance and represent changes in cell turnover of the syncytiotrophoblast.

Recurrent infection with human respiratory syncytial virus (RSV) is one of the most common causes for lower respiratory tract illness (LRI) in infants, the elderly, and immunocompromised individuals. Due to lack of vaccines and therapeutic interventions, medical care of acute RSV bronchiolitis is mostly limited to supportive measures. Thus, novel treatment options to control RSV infection are desperately required. The cationic host defence peptide human cathelicidin LL-37 possesses both microbicidal and immunomodulatory properties. This essential effector of the innate immune system holds potent antiviral activity against a variety of viruses, including influenza virus, and has been proposed as a promising candidate for antiviral drug development. Previous studies revealed that lower cathelicidin levels put RSV infected infants at risk for more severe RSV disease, while infection of lung epithelial cells induced cathelicidin up-regulation. These findings suggest that LL-37 might possess antiviral activity against RSV. However, its potential antiviral function on RSV remains to be elucidated. This thesis therefore aimed to evaluate the antiviral activity of cathelicidins against RSV, by assessing its relevance in vitro and in vivo and elucidating the underlying antiviral mechanism. Firstly, the antiviral effects of human cathelicidin LL-37 against RSV were addressed in vitro. Presence of LL-37 during infection potently reduced viral titres and protected cells against virus-associated cytopathic effects. Experiments revealed that only the core region of LL-37 holds antiviral activity against RSV. Antiviral effects were also observed for the murine LL-37 orthologue mCRAMP. Administration of LL-37 at different stages in the infection cycle provided evidence that LL-37 can be used preventatively, protecting against RSV infection by directly acting on both cells and viral particles. When given therapeutically, once an infection was established, LL-37 also limited viral spread. Next, the molecular mechanism mediating the peptide’s antiviral activity was investigated. It was demonstrated that LL-37 does not affect the interferon-mediated cellular antiviral immune response to RSV. Experiments established that LL-37 does not contribute to viral clearance by inducing epithelial cell death. Further mechanistic studies revealed that the peptide directly binds to RSV particles, destabilises the integrity of the viral envelope, and prevents adsorption of RSV to epithelial cells during the entry stage of infection. Finally, the in vivo relevance of LL-37 treatment and endogenous cathelicidin expression was examined, employing both murine and human model systems. It was established that LL-37 has protective antiviral effects against RSV in vivo. In contrast to the cell culture model, only co-administration of LL-37 and RSV, but not treatment prior or post infection, protects mice from clinical signs of infection. Levels of the murine LL-37 orthologue mCRAMP were increased in RSV infected lungs, pointing towards its importance in antiviral defence. In keeping with this, mCRAMP-deficient mice were more susceptible to RSV induced disease. Equally, individuals with low nasal LL-37 baseline levels that were experimentally challenged with RSV, were more susceptible to infection. This highlights the importance of endogenous cathelicidin expression to fight and control RSV infection. Overall, these results identify LL-37 as an important antiviral agent against RSV in vitro and in vivo, and emphasise the role of endogenous cathelicidins in the defence against this pathogen. Moreover, unravelling the underlying antiviral mechanism of LL-37 against RSV adds to our understanding of how CHDP act on enveloped viruses, thus supporting the development of new antiviral treatment options.

Thèse d'exercice : Médecine. Pédiatrie : Université de Nantes : 2004. / Bibliogr. f. 28-29 [23 réf.].

Thesis (M. Phil.)--University of Hong Kong, 2007. / Also available in print.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005. / Also available in print.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

The Mononegavirales order unifies the non-segmented negative sense viruses (nsNSVs), including Marburgvirus (MARV) of the Filoviridae family and respiratory syncytial virus (RSV) of the Pneumoviridae. The mechanism of action of these viruses and how they infect cells are very similar, especially when focusing on their polymerases, which are distinct from those of eukaryotes and therefore possible targets for antiviral drugs. nsNSVs utilize a RNA-dependent RNA polymerase to either replicate the viral RNA genome or transcribe it into positive sense mRNA. Despite this, these two viruses result in very different, but equally devastating, effects in humans. Whereas MARV virus often results in rare but fatal hemorrhagic fevers, RSV is a common seasonal virus that can result in long term negative effects to respiratory health. These negative effects on public health demand extensive research in these two fields and a need to develop new technology and methods in order to uncover the missing pieces of viral gene expression. Specifically, the development of a MARV minigenome system would allow for increased testing of this virus outside of the confines of the biosafety level 4 (BSL-4) setting. By replacing MARV genes with reporter genes, but retaining the characteristic leader, intergenic, and trailer regions of the genome, tests involving site directed mutagenesis would reveal new insights into the crucial genomic elements needed for successful gene expression. Coupled with the transfection of the minigenome with plasmids coding for the crucial MARV proteins, artificial changes to the genome would lead to the presence of absence of translated bioluminescent reporter proteins. Using these two viruses, this study attempted to find commonalities across families. Specifically, the goals of this research were twofold, to find the optimal ratio of MARV plasmids in the minigenome system to understand the effects of the stem-loop secondary structure of MARV mRNA transcripts as well as determine the tail length of the poly(A) tail of RSV mRNA transcripts using digestion and probing primers. Calculating the RSV poly(A) tail length would allow for further research into determining whether the MARV and RSV polymerase polyadenylates before or after it releases the transcript. Despite multiple failed attempts, transfections using pCAGGS plasmids and the eGFP monocistronic minigenome in a 6-well plate qualitatively demonstrated the need for pCAGGS-L plasmid concentration of 1000 ng/µl. Due to time constraints, the poly(A) tail length of the RSV NS-1 mRNA transcript could not be determined. Overall, this study focused on gaining new insights on the techniques and procedures necessary for conducting virus research in a biosafety level 2 (BSL-2) setting, as well as developing troubleshooting skills in approaching fail experiments.

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Background: Respiratory Syncytial Virus (RSV) is the leading cause of severe lower respiratory tract infection in infants and children worldwide. Early diagnosis of RSV infection is associated with shorter periods of hospitalisation and decreased mortality. Current point of care (PoC) tests for RSV is less sensitive than molecular methods. Reverse transcription loop-mediated isothermal amplification (RT-LAMP), is a novel method of nucleic acid detection which allows for rapid, robust amplification, and visual detection of infectious agents. Aim: The objective of this study was to develop a novel, rapid, and sensitive multiplex RSV RT-LAMP assay for PoC diagnosis of RSV A and B. Methods: Preparation of a quantitative RSV standard for assay optimisation was done using a rapid hypotonic burst recovery method of infective virus during sub-passaging, and a shell vial fluorescent focus assay for titration of culture-derived viral stock. We designed a single set of eight primers targeting the large polymerase gene of both RSV A and B, and developed a novel single-step multiplex RSV RT-LAMP assay, using an in-house reaction mix and the Rotor-Gene Q real-time thermocycler (Qiagen, Hilden, Germany). The metal ion indicator hydroxy naphtol blue (HNB) was added to the multiplex RSV RT-LAMP assay for visual detection of RSV. Results: The final optimised multiplex RSV RT-LAMP assay had an analytical detection sensitivity of <10 focus forming units (FFU) per reaction for both RSV A and B, with a mean time to positivity of 21.85 minutes (95% CI 19.2-24.5 minutes), compared to 90-120 minutes for conventional PCR. Evaluated against the Seeplex RV15 multiplex PCR (Seegene, Seoul, Korea) by testing 44 (22 RSV A/22 RSV B) nasopharyngeal specimens, the multiplex RSV RT-LAMP assay had a sensitivity of 100%, and a specificity of 100% when screened against nine common respiratory viruses. Visual detection of RSV using HNB as colorimetric reagent was equivalent to the analytical sensitivity (10 FFU/reaction) and specificity (100%) of the multiplex RSV RT-LAMP assay. Conclusion: Compared with conventional PCR, our novel single-step multiplex RSV RTLAMP assay had excellent sensitivity, specificity, and when combined with HNB dye could provide accurate visual diagnosis within 1 hour. We envisage that this multiplex RSV RTLAMP assay will be used for rapid and sensitive RSV detection at the PoC. / AFRIKAANSE OPSOMMING: Agtergrond: Respiratoriese Syncytial Virus (RSV) is die hoof oorsaak van erge laer lugweginfeksie in babas en kinders wêreldwyd. Vroeë diagnose van RSV infeksie word geassosieer met korter periodes van hospitalisasie en verlaagde mortaliteit. Huidige punt van sorg (PoC) toetse vir RSV is minder sensitief as molekulêre metodes. Omgekeerde transkripsie lus-gemedieerde isotermiese amplifisering (RT-LAMP), is 'n nuwe metode van nukleïensuur opsporing wat voorsiening maak vir vinnige, doeltreffende amplifisering, en visuele bevestiging van aansteeklike agente. Doel: Die doel van hierdie studie was om 'n nuwe, vinnige en sensitiewe multipleks RSV RTLAMP toets te ontwikkel wat PoC diagnose van RSV A en B in staat stel. Metodes: Voorbereiding van 'n kwantitatiewe RSV standaard vir toets optimisering is gedoen met behulp van 'n hipotoniese sel-lise metode van infektiewe virus tydens sub-kultuur, en 'n “shell-vial” kultuur en fluorosensie fokus toets vir titrasie van kultuur-geproduseerde virus voorraad. Ons het 'n enkele stel van agt inleiers ontwerp wat gebaseer is op die groot polimerase geen van beide RSV A en B, en 'n nuwe enkel-stap multipleks RSV RT-LAMP toets ontwikkel, met gebruik van 'n in-huis reaksie mengsel en die Rotor-Gene Q “real-time” thermocycler (Qiagen, Hilden, Duitsland). Die metaalioon aanwyser hidroksi naphtol blou (HNB) is bygevoeg in die multipleks RSV RT-LAMP toets vir visuele bevestiging van RSV. Resultate: Die finale geoptimiseerde multipleks RSV RT-LAMP toets het 'n analitiese sensitiwiteit van <10 fokus vormende eenhede (FFU) per reaksie vir beide RSV A en B gehad, met 'n gemiddelde tyd tot positiwiteit van 21.85 minute (95% CI 19.2-24.5 minute) , in vergelyking met 90-120 minute vir konvensionele PCR. Geëvalueer teen die Seeplex RV15 multipleks PCR (Seegene, Seoul, Korea) deur 44 (22 RSV A/22 RSV B) nasofaringeale monsters te toets, het die multipleks RSV RT-LAMP toets 'n sensitiwiteit van 100% getoon, en 'n spesifisiteit van 100% wanneer getoets teen nege algemene respiratoriese virusse. Visuele bevestiging van RSV met gebruik van HNB as kolorimetriese reagens was gelykstaande aan die analitiese sensitiwiteit (10 FFU/reaksie) en spesifisiteit (100%) van die multipleks RSV RT-LAMP toets. Gevolgtrekking: In vergelyking met konvensionele PCR, het ons nuwe enkel-stap multipleks RSV RT-LAMP toets uitstekende sensitiwiteit, spesifisiteit, en wanneer dit gekombineer word met HNB kleurstof kon dit akkurate visuele diagnose voorsien binne 1 uur. Ons verwag dat hierdie multipleks RSV RT-LAMP toets gebruik sal word vir vinnige en sensitiewe RSV bevestiging by die PoC.