>Magister Scientiae - MSc / Acute respiratory infections (ARI) continue to be the leading cause of acute illnesses worldwide and remain the most important cause of infant and young children mortality. Many viruses such as rhinoviruses, influenza viruses, parainfluenza viruses, respiratory syncytial viruses, adenoviruses and coronaviruses are deemed to be the etiological agents responsible for ARI’s in children. The recently discovered coronaviruses HCoV-HKU1 and HCoV-NL63 contribute significantly to the hospitalization of children with ARI’s. HCoV-NL63 was first identified in 2004, as the pathogen responsible for the hospitalization of a 7 month old child presenting with coryza, conjunctivitis and fever. Since then a significant amount of knowledge has been gained in the clinical spectrum on this virus, however HCoV-NL63 is still not well characterized on the molecular and proteomic level. This dissertation focuses on bringing about this characterization by cloning the HCoV-NL63 Nucleocapsid gene to be expressed in a bacterial system and transfecting the Nucleocapsid, Membrane and Envelope genes into a Mammalian cell culture system in order for its respective proteins to be expressed. With the use of Bioinformatic analytic tools certain characteristics of HCoV-NL63 Nucleocapsid, Membrane and Envelope proteins are able to be identified, as well as certain motifs and/or regions that are important in the functioning of these proteins. By comparing the results obtained for HCoV-NL63 N,M and E to other well studied coronavirus homologous will enlighten us on the potential role(s) of these proteins in determining HCoV-NL63 pathogenicity and infectivity. vi Although certain functions of these proteins can be deduced by the means of bioinformatics analysis, it is still imperative for it to be extensively characterized In Vitro. This will therefore form a fundamental step in the development of many other projects, which unfortunately fall outside the scope of this M.Sc thesis.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / The human coronavirus NL63 was discovered in 2004 by a team of researchers in Amsterdam. Since its discovery it has been shown to have worldwide spread and affects mainly children, aged 0-5 years old, the immunocompromised and the elderly. Infection with HCoV-NL63 commonly results in mild upper respiratory tract infections and presents as the common cold, with symptoms including fever, cough, sore throat and rhinorrhoea. Lower respiratory tract findings are less common but may develop into more serious complications including bronchiolitis, pneumonia and croup. The primary function of the HCoV-NL63 nucleocapsid (N) protein is the formation of theprotective ribonucleocapsid core. For this particle to assemble, the N-protein undergoes N-N dimerization and then interacts with viral RNA. Besides the primary structural role of the Nprotein, it is also understood to be involved in viral RNA transcription, translation and replication, including several other physiological functions. The N-protein is also highly antigenic and elicits a strong immune response in infected patients. For this reason the N-protein may serve as a target for the development of diagnostic assays. We have used bioinformatic analysis to analyze the HCoV-NL63 N-protein and compared it to coronavirus N-homologues. This bioinformatic analysis provided the data to generate recombinant clones for expression in a bacterial system. We constructed recombinant clones of the N-protein of SARS-CoV and HCoV-NL63 and synthesized truncated clones corresponding to the N- and C-terminal of the HCoV-NL63 N-protein. These heterologously expressed proteins will serve the basis for several post-expression studies including characterizing the immunogenic epitope of the N-protein as well identifying any antibody crossreactivity between coronavirus species. / South Africa

>Magister Scientiae - MSc / Human Coronaviruses (HCoVs) are found within the family Coronaviridae (genus, Coronavirus) and are enveloped, single-stranded, positive-sense RNA viruses. Infections of humans by coronaviruses are not normally associated with severe diseases. However, the identification of the coronavirus responsible for the outbreak of severe acute respiratory syndrome (SARS-CoV) showed that highly pathogenic coronaviruses can enter the human population. The SARS-CoV epidemic resulted in 8 422 cases with 916 deaths globally (case fatality rate: 10.9%). In 2004 a group 1 Coronavirus, designated Human Coronavirus NL63 (HCoV-NL63), was isolated from a 7 month old Dutch child suffering from bronchiolitis. In addition, HCoV-NL63 causes disease in children (detected in approximately 10% of respiratory tract infections), the elderly and the immunocompromised. This study was designed to express the full length nucleocapsid (N) proteins of HCoV-NL63 and SARS-CoV for antibody production in an animal model. The NL63-N/pFN2A and SARSN/ pFN2A plasmid constructs were used for this study. The presence of the insert on the Flexi ® vector was confirmed by restriction endonuclease digest and sequence verification. The sequenced chromatographs obtained from Inqaba Biotec were consistent with sequences from the NCBI Gen_Bank. Proteins were expressed in a KRX Escherichia coli bacterial system and analysed using 15% SDS-PAGE and Western Blotting. Thereafter, GST-tagged proteins were purified ith an affinity column purification system. Purified fusion proteins were subsequently cleaved with Pro-TEV Plus protease, separated on 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue R250. The viral fusion proteins were subsequently used to immunize Balbc mice in order to produce polyclonal antibodies. A direct ELISA was used to analyze and validate the production of polyclonal antibodies by the individual mice. This is a preliminary study for development of diagnostic tools for the detection of HCoV-NL63 from patient samples collected in the Western Cape. / South Africa

【研究緣起與問題】2003年春季，SARS在全球造成一陣轟動，同時也重創全球的經濟，如今SARS會不會再來？政府如何因應？政府如何監測疫情？如何防治疫情？如果SARS等新興及再浮現感染症 （Emerging and Re-emerging Infectious Diseases）出現時，負責全國傳染病防治任務的行政院衛生署疾病管制局是如何作好全國傳染病防治工作呢？中央及地方主管機關之溝通，如何共同消除指責與誤會，發揮團隊精神，如何共同作好全國傳染病防疫任務，爭取公眾對政府之瞭解並加以支持，同時防止任何可能之攻擊，才是全民之福祉。故以「全國傳染病通報系統」的績效，其所仰賴之溝通通路之問題研究，是本研究的動機。了解「傳染病通報管理系統(WEB版)」最大的問題及新興傳染病（如SARS）防治通報的問題及「傳染病通報管理系統」最需要改進之的地方，是本研究的目的。 【研究方法與資料】本研究採取量化與質化並重的方法，以網路問卷方式的進行問卷調查，問卷對象為全國使用傳染病通報管理系統之相關人員，問卷問題內容的設計主要的型式含有開放式問卷（Open –Ended Question）、封閉式問卷（Close-Ended Question）及混合型問卷（Mixed Question）三種。同時以九十二年針對新興傳染病SARS期間為例，深度訪談當時任職於中央與地方負責傳染病防治權責具代表性者五位主管進行訪談，瞭解受訪者對新興傳染病如SARS防治的問題及「傳染病通報管理系統」最需要改進之的地方，以彌補問卷調查法無法深入瞭解到問題之缺點。 【研究結論與建議】 一、根據筆者初步研究經問卷的實證調查之後，從問卷分析資料顯示，使用者使用電腦的時間愈久，對以新版為溝通工具滿意度愈低；而愈瞭解通報系統的使用者，其以新版為溝通工具滿意度愈高，愈滿意新版改進之電腦網路溝通效果。顯示新版的通報系統在設計的操作介面與程序上，沒有人性化，作業內容繁多、需輸入的項目、資料太繁瑣、版面太繁瑣等，反而徒增使用者的困擾；而愈瞭解通報系統的使用者，其對新版的滿意度越高，表示對於使用者在操作上的教育訓練仍須加強，才能發揮新版通報系統的成效。 二、從敘述統計分析中發現： 受訪者對「訊息異動通知作業流程滿意」的使用滿意度的平均數最高，平均數為2.8095表示：較為滿意。「通報作業流程滿意嗎」的使用滿意度的平均數最低，平均數為2.3934，受訪者表示：較為滿意。以新版為溝通工具整體溝通滿意情形，平均數為2.558滿意。 三、開放式問卷分析資料顯示，花太多時間、速度太慢、網路的頻寬過窄又不穩定或無法登錄、作業內容繁多、作業流程問題、資訊管理問題及功能增添與建議等部分。 四、根據筆者初步研究深度訪談之後，彙整中央主管機關及地方主管機關的訪談核心二部分的問題與建議。 五、研究建議： 綜合問卷調查及深度訪談結果，提出政策層面、組織溝通層面、管理層面、實務操作層面等之建議。 【關鍵字】傳染病通報系統、嚴重急性呼吸道症候群(severe acute respiratory syndrome, SARS)、組織溝通、溝通通路、行政院衛生署疾病管制局 / 【Background and Issues】 In the spring of 2003, SARS outbreaks devastated the whole world; they also heavily damaged the world economy. Will SARS come back? How can governments manage it? How can government monitor the epidemics? How can we control the infection? When emerging and re-emerging infectious diseases such as SARS appear, how can the Center for Disease Control of the Department of Health, an organization in charge of national disease control, plan and execute measures for the control of communicable diseases? Effective communication between the central and the local competent authorities to remove together blames and misunderstanding, to develop team spirit, to attain jointly the goal of disease control, to solicit understanding and support of the public to the government, and to prevent any likely attack are some of the considerations in improving the welfare of the public. The reason of the present study was to understand, through the achievements thus far of the National Communicable Disease Reporting System, issues involved in the communication channels. The purposes of the study were to understand the major problems of the Communicable Disease Reporting and Management System (the Web version), issues involved in the reporting of emerging infectious diseases, and areas of the Communicable Disease Reporting and Management System where improvement was most urgently needed. 【Method and Materials】 Both the quantitative and qualitative methods were used. Questionnaire interview was conducted through the web to persons using the Communicable Disease Reporting and Management System throughout the country. The questionnaire contained open-end questions, closed-end questions and questions of mixed types. At the same time, to understand in depth areas not properly covered by the questionnaire interview, five key persons responsible for disease control at the time of the SARS outbreaks in 2003 at the central and the local levels were interviewed to understand their attitudes toward the control of emerging infectious diseases such as SARS, and the improvement most urgently needed for the Communicable Disease Reporting and Management System. 【Results and Recommendations】 1. Preliminary findings from analysis of the questionnaire interview showed that users became less satisfied with the new version communication means the longer they were in use of computers. Users who knew more about the use of the reporting system were more satisfied with the new communication means, were at the same time, more satisfied with the communication effects of the improved version. These facts suggested that the new reporting system was less user-friendly in the designing of interface and procedures. The system required more work, more detailed inputs, and thus added additional burdens on the part of the users. The more the users understood the reporting system, their satisfaction was higher, indicating that, for the new version reporting system to function in full, training of the users in the use of the system should be strengthened. 2. From the descriptive analysis, it was found that the average score of user satisfaction on the “information change reporting procedures” was as high as 2.8095, suggesting that the users were fairly satisfied. The satisfaction score on “reporting procedures” was the lowest at 2.3934. The overall satisfaction score on the use of the new version as a communication means was 2.558. 3. Findings from the open-end questions gave the following recommendations: taking too much time, too slow, frequency of the web too narrow, unstable, and unable to load, too detailed, issues related to operational procedures, issues related to information management and additional functions, etc. 4. By analysis of the in-depth interview, some issues were raised and recommendations made to the central and the local competent authorities. 5. Through questionnaire survey and in-depth interview, some recommendations concerning policies, organizational communication, management and practical operation were made. 【Key Words】 Communicable Disease Reporting System, SARS (Severe Acute Respiratory Syndrome), organizational communication, communication channels, Center for Disease Control, the Department of Health

Antibiotic resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant enterococci (VRE), are an increasing burden on healthcare systems. Hospital acquired infections with these organisms leads to higher morbidity and mortality compared with the sensitive strains of the same species and both VRE and MRSA are on the rise worldwide including in Australian hospitals. Emerging community infectious diseases are also having an impact on hospitals. The Severe Acute Respiratory Syndrome virus (SARS Co-V) was noted for its propensity to spread throughout hospitals, and was contained largely through social distancing interventions including hospital isolation. A detailed understanding of the transmission of these and other emerging pathogens is crucial for their containment. The statistical inference and mathematical models used in this thesis aim to improve understanding of pathogen transmission by estimating the transmission rates of contagions and predicting the impact of interventions. Datasets used for these studies come from the Princess Alexandra Hospital in Brisbane, Australia and Shanxi province, mainland China. Epidemiological data on infection outbreaks are challenging to analyse due to the censored nature of infection transmission events. Most datasets record the time on symptom onset, but the transmission time is not observable. There are many ways of managing censored data, in this study we use Bayesian inference, with transmission times incorporated into the augmented dataset as latent variables. Hospital infection surveillance data is often much less detailed that data collected for epidemiological studies, often consisting of serial incidence or prevalence of patient colonisation with a resistant pathogen without individual patient event histories. Despite the lack of detailed data, transmission characteristics can be inferred from such a dataset using structured HiddenMarkovModels (HMMs). Each new transmission in an epidemic increases the infection pressure on those remaining susceptible, hence infection outbreak data are serially dependent. Statistical methods that assume independence of infection events are misleading and prone to over-estimating the impact of infection control interventions. Structured mathematical models that include transmission pressure are essential. Mathematical models can also give insights into the potential impact of interventions. The complex interaction of different infection control strategies, and their likely impact on transmission can be predicted using mathematical models. This dissertation uses modified or novel mathematical models that are specific to the pathogen and dataset being analysed. The first study estimates MRSA transmission in an Intensive Care Unit, using a structured four compartment model, Bayesian inference and a piecewise hazard methods. The model predicts the impact of interventions, such as changes to staff/patient ratios, ward size and decolonisation. A comparison of results of the stochastic and deterministic model is made and reason for differences given. The second study constructs a Hidden Markov Model to describe longitudinal data on weekly VRE prevalence. Transmission is assumed to be either from patient to patient cross-transmission or sporadic (independent of cross-transmission) and parameters for each mode of acquisition are estimated from the data. The third study develops a new model with a compartment representing an environmental reservoir. Parameters for the model are gathered from literature sources and the implications of the environmental reservoir are explored. The fourth study uses a modified Susceptible-Exposed-Infectious-Removed (SEIR) model to analyse data from a SARS outbreak in Shanxi province, China. Infectivity is determined before and after interventions as well as separately for hospitalised and community symptomatic SARS cases. Model diagnostics including sensitivity analysis, model comparison and bootstrapping are implemented.

<p> Virus-induced modulation of dendritic cell function is thought to be an effective mechanism for viral-immune evasion. The severe-acute respiratory syndrome coronavirus (SARS-CoV) has been shown to infected human myeloid dendritic cells (MDCs) and directly modulate the cellular cytokine production. The ability of other human coronaviruses to infect MDCs and impair cell immune function has not been assessed. </p> <p> This thesis describes the infection of human MDCs with coronavirus 229E, NL63, and OC43. 229E showed productive, but limited genomic replication, nucleocapsid protein synthesis and infectious progeny release in MDCs. 229E infection stimulated IFN-α, IL-6 and MCP-1 production in MDCs, but little to no IL-12, TNF-α, IL-8, IP-10, or RANTES . 229E-infected MDCs showed poor CD80 expression, down-regulated CD86 and HLA-DR expression and were poor stimulators of CD4+ T cell proliferation. In contrast to 229E, OC43 showed persistent and productive genomic replication, nucleocapsid protein synthesis and infectious progeny release in MDCs. OC43 infection stimulated IFN-α, IL-12, IP-10 and MCP-1 production in MDCs, but little to no TNF-α, IL-6, IL-8 or RANTES . The up-regulation of maturation molecules and CD4+ T cell stimulatory capacity in OC43-infected MDCs was donor cell-dependent. In contrast to 229E and OC43, NL63 infection of MDCs was non-productive, showing no viral genomic replication, protein production or infectious progeny release. NL63 infection stimulated strong cytokine (IFN-α, IL-12, TNF-β and IL-6) and chemokine (IL-8, IP-10, RANTES and MCP-1) responses in MDCs. NL63-infected MDCs showed up-regulated CD80, CD83, CD86 and HLA-DR expression and were efficient stimulators of CD4+ T cell proliferation. </p> <p> This study provides the first evidence that human coronaviruses other than SARSCo V can abrogate MDC immune effector function. It also provides the first side-by-side comparison of 229E, NL63 and OC43 and identifies the potential of 229E and OC43 to impair MDC cytokine production and T cell stimulation as a mechanism of immune response evasion. <p> / Thesis / Doctor of Philosophy (PhD)

40% of severe acute respiratory syndrome coronavirus two (SARS-CoV-2) severe cases develop acute kidney injury (AKI). Current treatment for renal complications limits financial and material resources available. To explore alternative treatments and accelerate research in case of future coronavirus outbreaks, a mouse model of coronavirus disease 2019-associated AKI (C19-AKI) would represent a critical biomedical research tool. The surrogate model of C19-AKI (SMC) developed consisted of angiotensin-converting enzyme two (ACE2) knockout (KO) mice receiving 400 ng/kg/min of angiotensin (Ang) II by osmotic minipump for eight days with a single injection of lipopolysaccharide (LPS; 10 mg/kg) on the seventh day of Ang II and euthanasia 24 hours after LPS. Similarly, to C19-AKI, the SMC exhibited albuminuria, elevated blood urea nitrogen, electrolyte imbalance, neutrophil infiltration, and upregulation of the G-coupled protein receptor (GPR)84 and pro-inflammatory and injury markers. GPR84 was found in bronchoalveolar lavage fluid neutrophils of coronavirus disease 2019 (COVID-19) patients, suggesting a potential implication of GPR84 in the disease. We hypothesised that GPR84 deletion or antagonism with GLPG-1205 could attenuate SMC’s indices of renal injury and inflammation. GLPG-1205 and GPR84 KO had no effects in the SMC model, as suggested by unchanged albuminuria, electrolytes, and markers expression. Interestingly, neutrophil infiltration was attenuated by GLPG-1205 only. The SMC is an interesting tool for therapeutic development for infections associated with renal injury, such as SARS-CoV-2. GPR84 role in the SMC needs to be further assessed.