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.

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.

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.

This retrospective cohort study examined risk factors associated with RSV-associated hospitalization (RSV-H) among infants born 32 to 35 weeks gestational age in Nova Scotia. Results were used to develop a clinical instrument (RSV-H scoring tool) that would discriminate between infants at high, moderate and low risk for RSV-H. Identifying the highest-risk infants, (using baseline information to predict RSV-H in the first year of life), would help target cost effective prophylaxis by Palivizumab (Pz), an expensive RSV-specific monoclonal antibody. Significant risk factors, determined by multivariate logistics, included infants born in December, January or February, passive household smoke exposure and household crowding. The scoring tool produced similar RSV-H post-test probabilities (3.1% pre-test probability) between risk groups (5.5% vs. 5.8%) and was unable to target highest risk infants. The tool could be used as an educational guideline for health professionals, outlining the importance of significant risk factors for RSV-H to parents and caregivers.

Thesis (Ph. D.)--University of Virginia, 2007. / Includes bibliographical references. Also available online through Digital Dissertations.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 86-100). Also available on the World Wide Web.