Macrophages have well-established roles in the primary response to pathogens and hold essential functions during innate and adaptive immunity. Under activation by different growth factors and cytokines, human monocytes have been shown to differentiate and polarize into two main types of macrophage, classically-activated macrophages (caMφ) and alternatively-activated macrophages (aaMφ), displaying distinct properties and phenotypes. For instance, caMφ secrete pro-inflammatory cytokines, whereas aaM secrete anti-inflammatory cytokines. Additionally, aaMφ displays stronger phagocytic ability and are equipped with different endosomal proteases. While it has been established that monocyte-derived macrophages can be infected by Influenza A virus, most studies utilized a macrophage population obtained by differentiation in the presence of autologous plasma. My research project aimed at systematically comparing susceptibility of the infection by Influenza A virus to the recently described caMφ and aaMφ. Here I show that monocytes cultured in presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon (IFN)-γ or in presence of macrophage colony-stimulating factor (M-CSF) and interleukin (IL)-4 or IL-10 can be differentiated into distinct populations. According to immunophenotyping results, a distinct expression profile was observed for Cluster of Differentiation (CD) 36, CD86, Mannose Receptor (MR or CD206), and Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN or CD209) among differentiated macrophages. Except for CD86 expression, my results were in accordance with previous reports and thus allowed me to classify all populations into caMφ (M1 macrophages), and aaMφ (M2a and M2c macrophages). I then assessed the susceptibility of the above mentioned macrophages to pandemic Influenza A/California/04/2009 H1N1 virus (CA04) infection. My results demonstrate a marked difference, caMφ showing low to moderate permissivity, whereas aaMφ – and in particular M2a macrophages – were consistently highly infected. In contrast, no difference was observed with Influenza A/WSN/1933 H1N1 virus (WSN/33) infection. Because sialic acids are regarded as the primary receptor for influenza virus, I investigated the cell surface distribution of sialic acids with α2-3 linkage (SAα-2,3) or α2-6 linkage (SAα-2,6) among the population of human macrophages. By using lectin staining with Maackia amurensis lectin (MAL) II and Sambucus nigra lectin (SNA), which bind sialic acids with α2-3 linkage (SAα-2,3) and α2-6 linkage (SAα-2,6) respectively, I found all the monocyte-derived macrophages exhibited a comparable expression of SAα-2,3 and SAα-2,6, which unlikely explain the differential susceptibility to infection by CA04. In addition to sialic acids, C-type lectins were also proposed to mediate entry of influenza viruses into macrophages. All macrophages expressed CD206 but only M2a expressed CD209. However assay aiming at interfering with CD209 binding (MAb blocking assay or EGTA treatment) did not inhibit pdmH1N1 infection. Surprisingly, infection in presence of EGTA, which is believed to reduce the functional ability of C-type lectins, exacerbated susceptibility of the macrophages. Altogether my results show that susceptibility to Influenza A virus infection of in vitro differentiated primary human macrophages is unlikely to rely on the sialic acid expression profile and is dependent on viral strain. Further studies are needed to understand what difference from caMφ and aaMφ – either phenotypic and/or biochemical – confer them distinct susceptibilities to some viral subtype/strain of Influenza A. / published_or_final_version / Pathology / Master / Master of Philosophy

BACKGROUND Influenza is an infectious disease that has significant public health impact due to its high prevalence and mortality. In early 2009, a novel influenza A(H1N1) virus emerged in Mexico and the USA, then rapidly spread worldwide and caused the first influenza pandemic of the 21st century. However, it is still controversial that whether seasonal influenza vaccine can provide a cross-protection against influenza A(H1N1) pdm09 infection. Since the 2009 pandemic occurred, numbers of studies focusing on this issue have been published, yet no confirmed conclusion was drawn. Therefore, further quantitative evaluation is needed to provide more reliable evidence. The objective of this study is to assess the cross-protection of seasonal influenza vaccination against 2009 pandemic A(H1N1) influenza illness, and explore the impact of seasonal influenza activities on this association. METHODS I followed the PRISMA statement and searched the PubMed, MEDLINE, Ovid Embase, The Cochrane Library databases, SCOPUS and CNKI. Randomized control trials, cohort studies, case-control studies assess the effect of seasonal influenza vaccine against influenza A (H1N1)pdm09 illness published in English and Chinese from 2009 to July 2013 were identified. The quality of included studies was assessed by the Jadad scale and the Newcastle-Ottawa Scale. I used the I2statistic, and Begg's funnel plot for assessment of heterogeneity and publication bias respectively. The software Review Manager 5.2 was used for generating the pooled effect with corresponding 95% confidence intervals and forest plots. Subgroup analysis was performed based on the study locations and previous circulating influenza viruses. RESULTS 20 studies were included in the meta-analyses. There is a non-significant 19% reduced risk of pandemic influenza illness in the countries combined data based on case-control studies(OR=0.81, 95% CI=0.60 to 1.08). While, for RCTs, a non-significant increase risk in seasonal influenza vaccinees was observed(RR=1.13, 95% CI=0.56 to 2.29). For the subgroup analysis, a significant 35% to 50% cross-protection was observed in South America and Europe, but an opposite result was observed in Canada(OR=1.44, 95% CI=0.83 to 2.50). Besides, the results indicate that there is no association between seasonal influenza vaccination and ILI. No publication bias was detected. CONCLUSIONS The findings partially support the hypothesis that seasonal vaccine may offer moderate cross-protection against laboratory-confirmed pandemic influenza A(H1N1) illness in general. Further immunological research is needed to understand the mechanism behind these findings. / published_or_final_version / Public Health / Master / Master of Public Health

The swine-origin H1N1 influenza A pandemic virus (A(H1N1)pdm09) was detected in the human population in March 2009. Due to its antigenic novelty, the majority of individuals were susceptible to the virus and the pandemic quickly disseminated around the globe. Rapid characterization of the epidemic was required in order to help inform interventions and determine the risk posed to public health. Widespread sampling and sequencing of virus isolates enabled early characterization of the virus using phylogenetic analysis and continued surveillance over the subsequent three years of global circulation. Throughout this thesis, Bayesian phylogenetic methods are employed to investigate how quickly evolutionary parameters can be accurately and precisely estimated from pandemic genome sequence data and explore how selection has acted across the A(H1N1)pdm09 genome over its period of transition to a seasonal influenza lineage. It is shown that accurate estimates of the evolutionary rate, date of emergence and initial exponential growth rate of the virus can be obtained with high precision from analysis of 100 genome sequences, thereby helping to characterize the virus just 2 months after the first cases were reported. In order to account for variation in growth rates of influenza epidemics between localized outbreaks around the globe, a hierarchical phylogenetic model is employed for analysis of pandemic and seasonal influenza data. The results suggest that the A(H1N1)pdm09 lineage spread more easily and with greater variation between populations during its first pandemic wave than either seasonal influenza lineage in previous seasons. The birth-death epidemiology model has been shown to provide more precise estimates of the basic reproductive number than the coalescent in analysis of HIV epidemic data. Analysis of pandemic influenza data carried out here suggests that the model assumptions are less applicable to influenza and in fact thebirth-death epidemiology model loses accuracy more rapidly than coalescent models as data increased during the pandemic. The effects of an increasingly immune global host population over the pandemic and subsequent influenza seasons were investigated using robust counting of substitutions across the genome. Results suggest that antigenic genes were under a greater selective pressure to evolve than internally expressed genes and the rate of non-synonymous substitution was highest across all segments immediately after emergence in the human population. Bayesian phylogenetics is increasingly being employed as an important tool for rapid characterization of novel infectious disease epidemics. As such, the work carried out here aims to determine the accuracy and applicability of existing evolutionary models with pandemic sequence data sampled over a range of temporal and spatial scales to help better inform similar analyses of future epidemics.

 The emergence of the pandemic H1N1 2009 virus of swine-origin and its transmission back to swine highlighted the need for global surveillance of swine influenza. Serology can help to address the epidemiological situation of influenza infection. Since typical serology tests such as hemagglutination inhibition or microneutralization assays are subtype and partially virus-lineage specific, it is important to select appropriate viral antigens for such studies. A poorly chosen panel of antigens will lead to underestimation of the seroprevalence. The choice of well-matched antigen is difficult if there is no prior virological surveillance in that area and even if there was virological surveillance data, transient infections may go undetected. Hence an universal influenza A type reactive serological test is needed. While such tests are available for poultry, there is little published data on the performance of these commercial influenza ELISA assays for serology on swine sera. In this study we evaluated 3 commercially available competitive ELISA assays, IDEXX? Influenza A Ab test, IDEXX? AI MultiS-Screen Ab Test and IDVet ID Screen? Influenza A Antibody Competition ELISA kit for detecting influenza type A reactive antibodies in swine. The virus antigens and the serum samples were obtained from a 14-year systematic abattoir-based virological and serological surveillance for swine influenza in southern China. The performance was evaluated by ROC curve and scatter plot, together with other statistical parameters including the Youden index to optimize the cut-off levels. Using the optimized cut-off levels, sensitivity and specificity of the IDEXX? Influenza A Ab test was 86% and 89% respectively; for IDEXX? AI MultiS-Screen Ab Test was 91% and 87% and for IDVet ID Screen? Influenza A was 95% and 79%, respectively. These findings help to provide different cut-off levels to maximize the sensitivity or specificity to suit different purposes. We found that the ELISA assay was useful in detecting serum samples that may be positive for influenza antibody but missed in the serology screening tests due to limitations in the chosen antigen panel. The ELISA assay maybe helpful in global swine influenza surveillance programs. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Background: The first influenza pandemic in the 21st century, the past 2009 influenza pandemic (pdmH1N1), was caused by a novel H1N1 influenza virus. The virus was first described in April 2009 and is now believed to emerge from re-assortment of bird, pig and human flu viruses. Although this pandemic was relatively mild compared to the past pandemics, better knowledge about its characteristics in transmission dynamics and severity is still of public health interest in order to better prepare for future pandemics. Data: Clinical surveillance data were obtained from eFlu database maintained by Hong Kong Hospital Authority. Information was extracted from all pdmH1N1 virologically confirmed infections (which were all symptomatic) about their dates of symptom onset, and, if applicable, dates of hospitalization, ICU admission and death. Serological data were obtained from various sources: 1) community cross-sectional serological survey; 2) convalescent serological data (from symptomatic and virologically confirmed infections); and 3) serological response kinetics data (from symptomatic and virologically confirmed infections). These serological data combined described serological responses against pdmH1N1 infections in the Hong Kong population from different aspects. Methods: I constructed an age-structured natural history model to mimic the pdmH1N1 transmission dynamics in Hong Kong. The transmission model was linked to hospitalization and serology in order to match the observed data. Based on all the data comprehensively, characteristic transmission parameters (basic reproductive number R0, mean generation time E(Tg), attack rates etc.) in the model were estimated using likelihood-based statistical inferences by Bayesian inference with Markov chain Monte Carlo (MCMC). Results: I estimated that R0 is 1.37 and E(Tg) is 2.16 days, which are both comparable to seasonal flu. Younger age groups <20 years were found to be more susceptible (2.72 times compared to 20-29 age group) to pdmH1N1 infection but older age groups 30-59 years were less susceptible (0.55 times). School closure reduced 0-12 year olds’ within-age-group transmission effectively during the reactive kindergarten and primary school closure from Jun 10 to Jul 9 by 93%. Summer holidays from Jun 10 to Aug 31 also reduced within-group transmission by 65% and 13% for 0-12 and 13-19 years olds respectively. Estimates of infection hospitalization probabilities ranged from 0.2% to 0.9% across age groups. I found that not all infected individuals would have serological response strong enough to be positive in serological test but younger age groups were more likely to have stronger serological response after infection. Conclusions: Clinical surveillance data have been used to estimate the transmission dynamics of pdmH1N1 in 2009. Here, I combined hospitalization surveillance data with serological data collected throughout the first pandemic wave (April to December 2009) from different sources, which could better characterize the transmission dynamics and severity of pdmH1N1 in Hong Kong. Although further validation is needed, serological surveillance should be considered as a supplementary alternative to clinical surveillance in influenza surveillance. / published_or_final_version / Community Medicine / Master / Master of Philosophy

學校停課在世界各國的流感大流行應對方案中常被列為一項社區緩疫措施，而這項措施亦在2009年H1N1流感大流行中被廣泛地使用。然而，這項緩疫措施經常被質疑是否恰當，原因是因為停課會對教育構成重大的影響，而且過往的流行病學硏究亦表示這項緩疫措施不一定有效。本論文硏究學校停課對2009年H1N1流感大流行在香港首5個月疫情中降低大流行流感傳播的效能。 / 在香港，在該大流行流感病毒於2009年4月在美國被發現後，香港政府實施了控疫措施(containment phase measures)，並開始對該流感大流行進行監測。為了判定大流行是否已在香港內蔓延，衛生防護中心設定了一個報告準則來讓本地醫生報告疑似大流行流感感染個案，並為每個懷疑個案作確診測試及為每個確診個案追溯感染源頭。當大流行流感在6月開始在香港內蔓延時，香港政府實施了緩疫措施(mitigation phase measures)。在緩疫措施底下，帶有流感病症的病人求診於指定流感診所和公共醫院急症室會被測試是否感染大流行流感，而停課措施亦在此時開始實行去減低大流行流感的傳播。停課措施一直維持至7月直至暑假開始，並經修改後於9月開學時繼續實行。在9月，鑑於已不再需要對流感大流行進行監測，對懷疑感染個案進行確診測試的政策止於該月下旬。確診個案中記錄了的病人資料，與及由學校停課和暑假所引起的學期變化，為這課題提供了一個理想硏究的機會。 / 在2009年的5月至9月，一共確診了27,687宗大流行流感個案。在確診個案中，所有個案都記錄了確診者的年歲和確診日期，而88%確診者提供了一個可定位的住宅地址。為了觀察學校停課的緩疫效果，本硏究定義了5個社會經濟年齡級別(socio-economic age classes) (當中包括有小學生和中學生)，並繪製了年齡級別與地域特定的疫情曲線(age-class-and-district-specific epidemic curves)。所有的疫情曲線在大流行流感在6月開始在香港蔓延後均穩步上升，而在屬於小學生和中學生的疫情曲線中能看到一個不尋常的上升出現在9月新學年開始時，意味著中小學生在學校的活動提升了大流行流感在他們之間的傳播。 / 先前，學校停課對減低2009年H1N1流感大流行在香港的傳播已被Wu et. al (2010a)進行了調查。透過使用一個具年齡結構的SIR模型(age-structured SIR model)來分析收集至8月27日的監測數據，該硏究表示流感大流行的傳播在暑假開始時減低了25%。在這研究中，我應用了Wu et. al (2010a)的方法來分析整個監測期間所收集的數據。在發現到該數學模型不能準確地擬合附加的監測數據後，我在該模型添加了兩個傳播特徵(當中包含兒童和成人之間的傳染在學校停課期間增加)去更準確地代表現實中的疫情。我的硏究顯示，學校停課雖然降低了兒童的感染率，但卻增加了成年人的感染率，令整體傳播在暑假開始時只減低了7.6%。這硏究結果表示，在將來的流感大流行中，封閉學校不大可能延遲流感大流行疫情至一個可令疫苗產生作用的程度，而且封閉學校可能會增加成人的感染率，從而有可能導致社會運作出現更混亂的情況。 / School closure is often included in national pandemic influenza response plans as a community mitigation measure and it was widely applied in Pandemic (H1N1) 2009. However, the appropriateness of this intervention is often questioned, as school closure causes major disruption to the education system and past epidemiological studies reveal this intervention is not necessarily effective. The present thesis evaluates the effect of school closure in mitigating transmission of Pandemic (H1N1) 2009 in Hong Kong in the initial 5 months of the pandemic. / In Hong Kong, following identification of the pandemic virus in US in April 2009, the government implemented containment phase measures and began surveillance on the pandemic. The Centre for Health Protection established a reporting criteria for doctors to report suspected cases of pandemic infection for laboratory confirmation, and the source of infection of confirmed cases was traced to determine if the pandemic was spreading locally. When local transmission of the pandemic began in June, the government began mitigation phase measures, in which patients with influenza-like- illness seeking treatment at designated flu clinics and public hospital emergency departments were tested for pandemic infection, and school closure was implemented for pandemic mitigation. The school closure policy lasted until summer holiday commenced in July, and was revised and continued in September when the new school season started. At the end of September, in view of pandemic surveillance was no longer useful, laboratory testing for suspected pandemic cases was halted. Patient demographic data collected from confirmed pandemic cases, together with temporal changes in school session induced by school closure and summer holiday, provided an ideal opportunity for investigation. / From May through September 2009, a total of 27,687 pandemic cases were confirmed, in which the age and confirmation date were recorded in all cases, and 88% provided a locatable residential address. To visualise the mitigative effect of school closure, 5 socio-economic age classes (which include primary and secondary school-aged children) were defined, and age-class-and-district-specific epidemic curves were constructed. All epidemic curves rose steadily after local transmission began in June, and an unusual upsurge in the epidemic curve of primary and secondary school-aged children is observed when schools resumed session in September, suggesting school session facilitated transmission amongst them. / Previously, the effect of school closure in mitigating Pandemic (H1N1) 2009 transmission in Hong Kong was investigated in Wu et al. (2010a). By analysing surveillance data collected as of 27 August with an age-structured susceptible- infectious-recovered (SIR) model, the study reported transmission was reduced by 25% when summer holiday commenced. In this study, I adapted the methodology in Wu et al. (2010a) to analyse data collected in the entire surveillance period. Upon observing the model fitted poorly to the additional data, I added 2 transmission features to the model (which include increased transmission between children and adults during school closure) to better represent the epidemic in reality. My analysis revealed that while school closure reduced incidence in children, it increased incidence in adults, leading to a reduction in overall transmission by only 7.6% when summer holiday started. The findings of this study suggest that school closure in a future influenza pandemic is unlikely to be able to delay the pandemic for vaccine to arrive in time, and that implementing this intervention may increase incidence in adults, which may lead to causing more disruption on the functioning of society. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chau, Kwan Long. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 148-154). / Abstracts also in Chinese. / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Influenza --- p.2 / Chapter 1.2 --- Public health response to pandemic influenza & School closure --- p.8 / Chapter 1.3 --- Pandemic (H1N1) 2009 --- p.13 / Chapter 1.4 --- Hong Kongs response to Pandemic (H1N1) 2009 --- p.17 / Chapter 1.5 --- Data and Research Objective --- p.24 / Chapter Chapter 2 --- Descriptive and Exploratory Analysis of Surveillance Data --- p.31 / Chapter 2.1 --- Introduction --- p.31 / Chapter 2.2 --- Methodology --- p.36 / Chapter 2.3 --- Results --- p.40 / Chapter 2.4 --- Discussion --- p.57 / Chapter Chapter 3 --- Evaluating the effect of School Closure by Modelling --- p.62 / Chapter 3.1 --- Introduction --- p.62 / Chapter 3.2 --- Methodology --- p.90 / Chapter 3.3 --- Results --- p.98 / Chapter 3.4 --- Discussion --- p.105 / Chapter Chapter 4 --- Discussion --- p.108 / Chapter 4.1 --- Study Findings --- p.108 / Chapter 4.2 --- Study Limitations --- p.109 / Chapter 4.3 --- Comments on using school closure in future influenza pandemics --- p.111 / Appendices --- p.116 / Bibliography --- p.148

abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Given that pandemic swine flu outbreak led to substantial admission in intensive care unit, extracorporeal membrane oxygenation has been increasingly applied to those who suffered from H1N1 infection induced acute respiratory distress syndrome. This review is going to evaluate the effectiveness of using ECMO based on five related observational studies. The result, discussion and policy implication in Hong Kong are discussed. Since the ECMO system has been technological improved in recent years, there are less complications when applying ECMO. In view of evidence of reviewed studies, application of ECMO in Hong Kong can be considered as cost effective. And since only a few hospitals in Hong Kong can offer ECMO application, retrieval teams are needed to ensure safety transfer between hospitals. / published_or_final_version / Public Health / Master / Master of Public Health

Mannose-binding lectin (MBL) functions as pattern recognition molecule to mediate first-line host defense against invading pathogens. Although MBL is well-known for its anti-bacterial action, its role towards virus infection is less comprehensively understood. In 2009, the pandemic H1N1 2009 (pdmH1N1) influenza A virus caused more than 18,000 deaths worldwide and is still circulating in human community as a seasonal strain. In this study, the role of MBL in pdmH1N1 infection was investigated. Using in vitro microtiter capture assay, MBL was found to bind to pdmH1N1 virus via its carbohydrate recognition domain. Under transmission electron microscope (TEM), MBL was clearly visible on the surface of pdmH1N1 virus. By infecting C57B6/J wild-type (WT) and MBL knockout (KO) mice with a sub-lethal dose of pdmH1N1 virus, WT mice displayed greater weight loss and more severe lung damage than MBL KO mice. Using flow cytometry-based profiling analysis of the lung homogenates isolated from infected mice, a variety of proinflammatory cytokines and chemokines were found to be significantly up-regulated. These results indicate that the presence of MBL can cause excess proinflammatory cytokine production and result in a more severe pdmH1N1 infection. To provide physiologically relevant insight into the immunomodulating role of MBL, the investigation was further extended to the use of human cell line model. Infection of A549 cells, which is a human lung epithelial cell line, with MBL-bound pdmH1N1 virus elevated the production of MCP1, RANTES and IL-8 significantly more than unbound pdmH1N1 infection. The increased production of chemokines also enhanced recruitment of monocytes as demonstrated by transwell migration assay. Interestingly, MBL did not affect viral entry or replication kinetics. TEM and confocal imaging revealed the presence of MBL-bound pdmH1N1 inside infected A549 cells, suggesting that the endocytosed MBL may interact with intracellular components to promote the release of cytokines and chemokines. To this end, expressions of Toll-like receptors were examined (TLR3, TLR7, TLR8 and TLR9) and found that TLR3 expression was dramatically enhanced upon pdmH1N1 infection. Interestingly, in MBL-bound pdmH1N1 infection, TLR3 mRNA and protein expression was significantly higher than unbound pdmH1N1 infection in A549 cells. In addition, the NF-κB signaling was further activated in the presence of MBL-bound pdmH1N1. A novel physical interaction between MBL and TLR3 was also delineated as evidenced by MBL’s capability to bind to TLR3 in vitro; and their colocalization in the endosomes of the infected A549 cells. In summary, MBL can bind to pdmH1N1 virus but fails to inhibit its infection in human lung epithelial cell line. Upon pdmH1N1 infection, MBL is internalized with the virus into the cell, where it may associate with TLR3 to further amplify the NF-κB signaling and augment the cytokine production in the human lung epithelial cells. The present findings advocate the adverse immunomodulating role of MBL during pdmH1N1 infection. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

During the 2009 H1N1 influenza pandemic, public health nurses (PHNs) were called upon to protect their communities against a deadly influenza virus. Currently, there appears to be no literature that describes the experience of Canadian PHNs responding to the first influenza pandemic of the 21st century. A qualitative research study was conducted and the data were analyzed by using content analysis. Thirteen nurses were interviewed from an Urban, Rural and Northern health region in Manitoba. Focus groups were conducted for the Urban and Rural nurses while Northern nurses were interviewed by telephone. Communication and dissemination of information, personal and professional challenges, personal face of the pandemic, regional support and lessons learned were themes generated from the data analysis. Communication and information flow was a major theme that impacted the overall PHNs’ response experience. Practice, administration, research and education implications and the limitations of the study are presented in the study.