Thesis (M. Med. Sc.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 40-46).

Thesis (M. Med. Sc.)--University of Hong Kong, 1983.

published_or_final_version / Microbiology / Master / Master of Medical Sciences

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

During the 1997 Hong Kong ‘bird flu’ incident, three subtypes of influenza viruses, including H5N1, H9N2 and H6N1, were co-circulated at the live-poultry markets. Genetic analyses revealed that all these viruses shared the same internal gene complex and might have been all involved in generation of the HK/97-like H5N1 virus. Subsequent epidemiological and genetic studies found that both H6N1 and H9N2 viruses became established and prevalent in minor poultry in the region. However, the genesis pathway for each of these viruses has not been defined. It is also unclear about these three subtypes further interact with each other and evolve in the field, along with the emerging reassortant variants. To address these questions, H6 subtype of avian influenza viruses isolated from terrestrial minor poultry from 2000 to 2005, and from 2006 to 2007 in our influenza surveillance in southern china has been genetically and antigenically analyzed in this study. Genetic and phylogenetic analyses of representative strains indicated that all H6N1 isolates from 2000 to 2007 had W312-like hemagglutinin and neuraminidase genes. These H6N1 viruses have become established in the minor poultry, mainly in quail and chukar, in this region. However, phylogenetic analyses revealed that the internal genes of the H6N1 virus lineage were derived from multiple origins with different evolutionary pathways. Evolution analyses of different gene segments of H6N1 viruses revealed imbalance dynamic evolutionary rates between surface genes and internal genes, which suggests that this virus lineage was more likely a descendant of the HK/97-like H5N1, rather than its precursor virus. Similar to what have been observed in the H5N1 and H9N2 virus lineages, the internal gene complex of the H6N1 viruses was found to undergo extensive reassortment. Many novel internal gene segments of H6N1 viruses were first recognized in the reassortant H9N2 virus particles, suggesting that the gene flow is likely from H9N2 to H6N1. The co-circulation of different virus lineages in southern China has greatly increased the genetic diversity of influenza viruses in this region. Analyses of the dynamics of different H6N1 reassortant variants also showed that some of them became persistent, but others were transient in the field. The increasingly diversified H6N1 and other subtypes of viruses will naturally increase the opportunity of interspecies transmission and dissemination, and may pose renewal threat for public health. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

The outbreaks of human influenza caused by highly pathogenic avian influenza H5N1 virus have attracted a lot of attention and public concern. Effective and universal vaccines may be the best means for prevention and control of the influenza. Taking into account that viral clearance and recovery from influenza A virus infection have been demonstrated to be correlated to specific cytoxic T lymphocyte (CTL) instead of neutralizing antibodies, it is important to develop effective vaccines which are capable of inducing not only neutralizing antibody but also CTL responses. Furthermore, T cell epitopes are usually more conserved than neutralizing epitopes. However, rare information concerning human T cell epitopes specific to H5N1 virus has been reported so far. This study was designed to test our hypothesis that novel and potent human CTL and Th epitopes specific to NP protein of H5N1 virus may be identified in vaccinated and/or infected HLA-A2/DR1 transgenic mice (SURE/L1), while protective epitopes may be further defined from the identified T cell epitopes in the mice challenged with lethal dose of the virus. We used SURE/L1 mouse model because it contains HLA-A2 (*0201) and -DR1 (*0701), both are the second highest frequency of HLA class I and II in Chinese. Since the NP gene is relatively conserved among different clades or strains of H5N1 virus, we selected viral protein NP as the target. Furthermore, we screened the T cell epitopes in splenocytes not only from vaccinated mice but also from survived mice infected with gradually increased dose of H5N1 virus, because the T cell epitopes identified in both vaccinated and infected mice or in infected mice alone might have higher potential to be protective epitopes. In this study, a novel HLA-DR1 (class II) restricted T cell epitope NP368-382, NPII-7, was identified in both vaccinated and infected mice. Two doses of NPII-7 peptide boosting in the mice induced very strong Th1 and CTL responses but no NP specific antibody responses. The vaccination of additional 2 doses of NPII-7 also provided partial protection against lethal challenge of H5N1 virus in the mice, whereas NP DNA vaccination alone did not show any protective effect. The protective effect may be attributed to the strong Th1 and CTL responses induced by the NPII-7 vaccination, because both NP DNA and NPII-7 vaccinations could not induce neutralizing antibody response. Notably, a HLA class II restricted peptide, NPII-7, may induce not only Th1 responses but also more strong HLA class I restricted T cell (CTL) responses. It may probably due to that the HLA-DR1 restricted T cell epitope (NENMEAMDSNTLELR) contained the full sequence of a reported HLA-A2 restricted CTL epitope (AMDSNTLEL), named NP-17 in this study. Although it needs to be further defined whether this novel epitope is really a HLA-DR1 restricted T cell epitope, or it shares the activity of HLA-A2 restricted T cell epitope, or it is just an alternate HLA-A2 restricted T cell epitope, this study has identified a novel T cell epitope and proved that it is a protective T cell epitope. / published_or_final_version / Microbiology / Master / Master of Philosophy

Background: Influenza viruses cause substantial mortality and morbidity both worldwide and in Hong Kong. Furthermore, the possible emergence of future influenza pandemics remains a major threat to public health. Some studies have estimated that one third of all influenza transmission occurs in households. Household studies have been an important means of studying influenza transmissions and evaluating the efficacy of influenza control measures including vaccination, antiviral therapy and prophylaxis and non-pharmaceutical interventions. Household studies of influenza can be categorized as pertaining to one of two designs: household cohort and case-ascertained. In household cohort studies households are recruited before the start of an influenza season and then monitored during the influenza season for influenza infection. In case-ascertained studies a household is enrolled once influenza infection is identified in a household member. Objectives: This thesis comprises of two parts. The objective of the first part is to evaluate the resource efficiency of different designs for conducting household studies. The objective of the second part is to estimate community and household transmission parameters during the 2009 A(H1N1) pandemic in Hong Kong. Methods: Monte Carlo simulation parameterized with data from influenza studies in Hong Kong was used to compare the resource efficiency of competing study designs evaluating the efficacy of an influenza control intervention. Approaches to ascertaining infections in different types of studies, and their implications for resource efficiency were compared. With regard to the second part, extended Longini-Koopman models within a Bayesian framework were used on data from a Hong Kong household cohort study conducted from December 2008 to October 2009. Household and community transmission parameters were estimated by age-groups for two seasonal influenza strains circulating in the winter of 2008-09 and two seasonal and one pandemic strain circulating in the summer of 2009. Results: Simulations showed that RT-PCR outperformed both serology and self-report of symptoms as a resource efficient means of identifying influenza in household studies. Identification of influenza using self-report of symptomatology performed particularly poorly in terms of resource efficiency due to its low sensitivity and specificity when compared to laboratory methods. Case-ascertained studies appeared more resource efficient than cohort studies but the results were sensitive to the choice of parameter values particularly the serial interval of influenza. In statistical analyses of household data during the winter of 2008-09, it was found that transmissibility of seasonal influenza strains were similar to those previously reported in the literature. Analysis also showed for the summer 2009 the estimates of household transmissibility were similar for seasonal A(H3N2) and pandemic A(H1N1) especially after taking into account that some individuals were likely immune to infection. Conclusions: Careful consideration of study design can ensure that studies are resource efficient and have sufficient statistical power. Data from a household study suggested that during 2009 seasonal and pandemic influenza had similar transmission patterns. / published_or_final_version / Community Medicine / Doctoral / Doctor of Philosophy

Although several risk factors for severe influenza infection have been identified in previous studies, many patients having multiple risk factors only developed mild symptoms while many healthy young patients developed severe complications when infected with A(H1N1)pdm09. Thus, there are still undiscovered factors that affect the progression and severity of influenza. The early innate immune response may be critical in determining the disease progression. Non-neutralizing antibodies existed in the early stage of infection may contribute to the outcome of the disease. In this study, the association of disease severity with the titre and avidity of non-neutralizing antibodies in early stage of influenza infection was investigated. It has been shown that the titre of non-neutralizing antibody was higher in more severe patients in the early stage of infection. Higher antibody avidity was also found to be associated with more severe disease independently. These findings tend to support the view that antigenic drift leads to an excessive production of pro-inflammatory non-neutralizing antibodies in the patients and associated with severe outcome. Since patients with more severe disease tend to have a delayed clearance of the virus and allow more transmission, the antigenically shifted or drifted influenza virus may gradually become predominant in human population. This idea suggested that the predominance of influenza virus with NA-H275Y mutation in 2007-2008 was contributed by the co-existing, fitness restoring secondary adaptive mutation in HA. NA-H275Y was identified in previous studies to be the mutation encoding for the influenza virus to resist against oseltamivir but would also change the property of NA as a result of compromised viral fitness. Therefore, influenza virus carrying NA-H275Y is unlikely to emerge and spread in human population. However, NA-H275Y mutated strains of influenza virus emerged and spread globally in the influenza season of 2007-2008 and quickly become the predominant strain in 2008. Previous study found NA-R222Q and NA-V234M were the mutations responsible for restoring the viral fitness in oseltamivir resistant clinical isolates. Still, this cannot fully explain the predominance of the resistant strains over the susceptible strains. Therefore secondary adaptive mutation in HA was believed to be present and cause antigenic change to the resistant strains of influenza. In this study, mutual information analysis and HA structural analysis were conducted to screen out HA-A189T and HA-Y94H to be the candidates co-exist with NA-H275Y and possibly critical for antigenic changes. This study further suggested that HA-Y94H mutation leads to a change of antigenic property of the virus by examining the antigenicity and growth kinetics of the recombinant viruses carrying the selected HA mutations. HA-94 may be critical for determining both the receptor binding property and antigenic property of the virus. Review on the evolution of seasonal influenza viruses from 2005 to 2008 suggested that the emergence of HA-Y94H mutation may enhance the presence of NA-H275Y and helps the viruses carrying NA-H275Y to spread and dominate over the oseltamivir susceptible strains during 2007 and 2008. / published_or_final_version / Microbiology / Master / Master of Philosophy

The influenza polymerase is a complex of three subunits, polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1) and polymerase acidic protein (PA). It associates with the viral RNA segment and nucleoprotein (NP) to form a viral ribonucleoprotein (vRNP) complex which is important for transcription and replication of the viral genome. Concurrently, the previous three influenza pandemics viruses contain reassorted vRNP of different origins. This leads to the aim of study to investigate the role of polymerase in the pandemic viruses. By reconstitution of vRNPs in human cells, it was demonstrated that vRNPs of H2N2 and H3N2 pandemic viruses had higher polymerase activity than the H2N2 seasonal viruses in-between them. The recombinant virus with H2N2 pandemic vRNP also showed faster growth kinetics in the early stage of viral replication and better adaptability to the selective environment with neuraminidase inhibitor than the recombinant virus with H2N2 seasonal vRNP, which had a lower polymerase activity. Reconstitution of chimeric vRNPs of H2N2 pandemic and seasonal viruses revealed that PB2, PB1 and PA were responsible for the difference in polymerase activity between them. Five residues, one in PB2, three in PB1 and one in PA were identified to be significant for the polymerase activity change. These polymerase subunits and residues may act as part of the determining factors for the H2N2 pandemic virus. Furthermore, PB2-627 has been shown to have stringent host specificity and affect polymerase activity and viral replication. Recombinant viruses in mammalian and avian cells with random mutation were generated at this position. It showed that the amino acids at this position are not restricted to those appear in the nature for generating viable viruses. It was also observed that the avian-derived viruses generally had lower polymerase activity and reduced growth kinetics in mammalian cells, while part of the mammalian-derived viruses had lower polymerase activity and reduced growth kinetics in avian cells. This consolidated the role of PB2-627 on host specificity and demonstrated the possibility of some novel amino acids for this position, which may play a role in the future influenza pandemic. The 2009 H1N1 pandemic virus contains a reassorted vRNP with subunits of avian, human and swine origins. This prompts me to compare the polymerase activity of all the 81 possible combinations of chimeric vRNPs of three different origins. The results were statistically analyzed and several single subunit factors and interactions between vRNP subunits were identified to significantly affect the polymerase activity. In order to reduce the effort and resources required, a fractional factorial design of 27 experimental runs was developed to substitute the 81-combination full factorial design for identifying the significant single subunit factors that affect the polymerase activity. Overall, this study identified some factors that may contribute to a pandemic virus and allows us to have better understanding of the role of polymerase in a pandemic virus. These findings may contribute to evaluating the pandemic potential of the novel virus that emerges or may emerge in the nature and enhances the preparedness towards the next pandemic influenza. / published_or_final_version / Public Health / Doctoral / Doctor of Philosophy

Pregnant women infected with influenza virus are more likely to experience severe complications compared with their non-pregnant peers. Yet influenza vaccine uptake is low among pregnant women. The purpose of this study was to assess the prevalence and predictors of seasonal influenza vaccine uptake among pregnant women in Hong Kong. Using a multi-centre cross-sectional design, we recruited 2,822 new mothers during their immediate postpartum stay at all eight public obstetric hospitals over a three-month period from April through June 2011. We assessed their antenatal maternal influenza vaccination status as well as health beliefs and perceptions toward influenza and influenza vaccination. Bivariable and multivariable logistic regression was used to identify the predictors of vaccination uptake. Only 49 (1.7%; 95% CI 1.3% to 2.3%) participants were vaccinated during pregnancy. Fears that the vaccine would harm their foetuses or themselves were the most common reasons for not being vaccinated. Being aware of vaccination recommendations (OR=2.69; 95% CI 1.06, 6.82), being advised by a health care provider (HCP) to be vaccinated (OR=6.30; 95% CI 3.19, 12.46), a history of influenza vaccination (OR=2.47; 95% CI 1.25, 4.91), perceived susceptibility to influenza infection (OR=3.67; 95% CI 1.64, 8.22), and perceived benefits of influenza vaccination (OR=9.98; 95% CI 3.79, 26.24) were all independently associated with vaccination. Perceived barriers to vaccination (OR=0.17; 95% CI 0.07, 0.40) were strongly associated with failure to vaccinate. A qualitative descriptive design was also used to explore a broad spectrum of health knowledge and beliefs of participants regarding influenza infection and influenza vaccination during pregnancy. An interview guide was developed based on the Health Belief Model. A sub-sample of participants who completed the quantitative study were invited to take part in the qualitative interviews. A total of 32 postpartum women were interviewed and only two had been vaccinated during pregnancy. Following thematic analysis, three themes emerged that further highlighted the pregnant women’s perceptions toward influenza vaccine and their decision-making process, perceived risk of influenza infection, perceived risk of an influenza vaccine, and decision-making cues. Overall, participants held negative impressions about influenza vaccination during pregnancy. This could be because of misconceptions and underestimation of the threats of influenza infection to themselves and their foetuses. They were also confused about the safety and efficacy of the influenza vaccine. Participants were confused about the differences between preventive strategies and treatment for influenza and HCPs did not offer or recommend vaccination. Because of negative media reports about the pros and cons of vaccination, participants were hesitant to receive the vaccine. Nevertheless, findings suggested that motivating forces for vaccine acceptance were a high prevalence of circulating influenza infection during their pregnancy and HCP recommendations and reassurances that the vaccination was safe, effective, and beneficial for the foetus. Vaccination promotion strategies need to focus on encouraging HCPs to discuss vaccination with their pregnant clients and provide accurate and unbiased information about the risks of influenza infection and the benefits of vaccination. / published_or_final_version / Nursing Studies / Doctoral / Doctor of Nursing