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A variety of factors are thought to contribute to the pathogenesis of avian influenza (AI) viruses. Three of these factors: host, immunity and virus strain, were studied. Specifically, tissue tropism in hosts usually regarded as resistant to infection with HP AI viruses; the effect of prior immunity on infection; the effect of age on disease outcome and the genetic factors of the virus were studied. The ability of "Asian- lineage" A/turkey/Turkeyll/2005 (H5Nl) HPAI virus (clade 2.2) virus to produce disease and mortality in Pekin ducks was affected by the age at infection. In 4- week-old Pekin ducks, infection resulted in systemic infection, severe neurological signs and 100% mortality. Viral shedding was primarily from the oropharyngeal route. Only mild clinical signs and no mortality was observed in 12-week-old ducks infected with the same dose of this virus. Clinical signs and mortality rates in 8- week-old ducks were similar to those observed in 4-week-old ducks, but viral shedding and virus loads in tissues were shown to be lower, and generally absent in brain and heart tissues in both age groups of older ducks. Prior infection of 3-week- old ducks with a low pathogenicity AI (LP AI) H5N2 virus and subsequent challenge at 7- and 16-weeks-old with A/turkey/Turkey/1/2005 HPAI H5Nl was shown to decrease the severity of clinical signs and the amount of virus shed, although in this particular study mortality was observed in neither prior infected or naive birds on challenge. Viral genetic markers for virulence of HP AI viruses in chickens were studied in two genetically similar HP AI H5Nl viral clones - A/turkey/England/50-92/l 991 2L and A/turkey/England/87-9211991 LDP3, which displayed high and low virulence in intravenous pathogenicity (IVPI) tests with indices of 1.77 and 0 respectively. Ten amino acid differences in five genes - PB1, PB2, PA, HA and NP, were identified between these two isolates. Nine of these differences, the exception being the one in the NP gene, were shown to contribute to the high virulence of the 2L clone since they were induced in the low virulence LDP3 clone after intracerebral passage in day-old chicks and led to a change in phenotype to a high IVPI score. Two mutations in PBl unique to LDP3, located in a known functional domain involved in the binding ofPBl to both PB2 and vRNA, were shown to reduce 2L polymerase function significantly in chicken cells when measured by an in vitro polymerase activity assay. This finding highlights the important contribution of polymerase function to the virulence of AI viruses. The results from the present study collectively highlight the complex polygenic nature of HP AI virus infections in avian hosts.

The possible emergence of a highly pathogenic avian influenza virus (HPAI) which can cross the species barrier to infect new hosts, avian or mammalian, has become a serious threat and public concern. The timely diagnosis and isolation of such viruses is crucial for outbreak control and the development of drug and vaccine responses. Modelling suggests that the next outbreak is likely to occur in rural area where the practical difficulties of management would be enormous. Therefore, the development of rapid and simple diagnostic tests to identify the index case of HPAI infection, under field conditions, is essential. Currently available rapid diagnostic tests for influenza A viruses detection suffer from low sensitivity and a lack of the ability to identify potential pandemic subtypes. This study sought to develop a rapid diagnostic test for type A influenza viruses as well as investigate the potential for identification of pandemic subtypes HS, H7, and H9. The aim was to demonstrate that tests could be developed in-house that were more sensitive, cost- effective, and easy to use than the existing commercial products, notably by using immunomagnetic beads. To do this, antigens and polyclonal antibodies necessary for using in the detection of influenza A viruses as well as potential pandemic subtype HS, H7, and H9 infections were prepared starting from the sequence analysis through to the purification process. The material generated was shown to be highly selective for their target analytes and did not cross-react with antigens or antibodies from other related respiratory diseases. In particular, the NP protein showed an equivalent performance to the existing commercial rapid test products: ClVTEST (HIPRA, Spain) and FlockChek (lDEXX, USA), in identifying type A influenza virus antibodies. Moreover, when fabricated with magnetic bead particles, an NP-immunomagnetic bead assay was shown to improve the analytical sensitivity in detecting influenza A virus protein ~320 times when compared with a conventional ELlSA. Although subtype specifi c assays for HS, H7, and H9 remain challenging due to their highly variable antigenic structure, immunomagnetic bead assays succeeded in demonstrating an improvement in not only the analytical sensitivity but also the specificity of the H7 HA subtype diagnosis when compared with a standard ELlSA. Therefore, immunomagnetic bead assay have the potential to be further developed and used as rapid test to improve the sensitivity of influenza virus diagnosis under field conditions.

Neutralising antibodies and antigen-specific B cells are important for protection against influenza A virus. However, the antigenic evolution of influenza A viruses has made a continuing challenge to the design of vaccine and the public health. The ability to generate cross-reactive response against influenza remains unclear in human. It is important to explore the antibody and B cell repertoire at single cell level. The pandemic H1N1 and seasonal influenza vaccine induced robust antibody response in adults. However, pre- or co-vaccination with the seasonal vaccine led to a significantly reduced antibody response to pandemic H1N1 virus. Whether this interference has impact on subsequent infection rates remains undetermined. There observed substantial cross-reactive antibody response upon vaccination, as measured by HI, MN and B cell ELISpot assays. The antibody recognizing conserved proteins could be the main component of cross-reactivity against influenza A strains and subtypes. A significant expansion of influenza-specific MBC was observed after infection. Crossreactive response was also noted in the MBC response. Importantly, a robust early-phase ASC response was detected in the peripheral blood upon influenza vaccination or infection. The size of ASC response significantly correlated with serum HI, MN and anti-HA IgG titre three weeks after vaccination. The sequence analysis revealed that early-phase ASC accumulated high level of somatic mutations on Ig variable region and affinity maturation, as well as anti-influenza mAb, which suggested their origin from pre-existing MBC. Eight anti-influenza mAb were made from early-phase ASC, including one high-titre virus-neutralising HA1-specific, two other HA1-specific, one cross-reactive HA2-specific, and four cross-reactive NP-specific antibodies, indicating of the broad diversity of ASC repertoire. In conclusion, this study demonstrated the properties of antibody and B cell responses to influenza A virus at serological, cellular and sequence level. The virus-neutralising and cross-reactive mAb derived from ASC could have therapeutic potential and their analysis might direct the vaccine design in the near future.

A range of simulations have been conducted to investigate the behaviour of a diverse set of complex biological membrane systems. The processes of interest have required simulations over extended time and length scales, but without sacrifice of molecular detail. For this reason, the primary technique used has been coarse-grained molecular dynamics (CG MD) simulations, in which small groups of atoms are combined into lower-resolution CG particles. The increased computational efficiency of this technique has allowed simulations with time scales of microseconds, and length scales of hundreds of nm. The membrane-permeabilizing action of the antimicrobial peptide maculatin 1.1 was investigated. This short α-helical peptide is thought to kill bacteria by permeabilizing the plasma membrane, but the exact mechanism has not been confirmed. Multiscale (CG and atomistic) simulations show that maculatin can insert into membranes to form disordered, water-permeable aggregates, while CG simulations of large numbers of peptides resulted in substantial deformation of lipid vesicles. The simulations imply that both pore-forming and lytic mechanisms are available to maculatin 1.1, and that the predominance of either depends on conditions such as peptide concentration and membrane composition. A generalized study of membrane protein aggregation was conducted via CG simulations of lipid bilayers containing multiple copies of model transmembrane proteins: either α-helical bundles or β-barrels. By varying the lipid tail length and the membrane type (planar bilayer or spherical vesicle), the simulations display protein aggregation ranging from negligible to extensive; they show how this biologically important process is modulated by hydrophobic mismatch, membrane curvature, and the structural class or orientation of the protein. The association of influenza hemagglutinin (HA) with putative lipid rafts was investigated by simulating aggregates of HA in a domain-forming membrane. The CG MD study addressed an important limitation of model membrane experiments by investigating the influence of high local protein concentration on membrane phase behaviour. The simulations showed attenuated diffusion of unsaturated lipids within HA aggregates, leading to spontaneous accumulation of raft-type lipids (saturated lipids and cholesterol). A CG model of the entire influenza viral envelope was constructed in realistic dimensions, comprising the three types of viral envelope protein (HA, neuraminidase and M2) inserted into a large lipid vesicle. The study represents one of the largest near-atomistic simulations of a biological membrane to date. It shows how the high concentration of proteins found in the viral envelope can attenuate formation of lipid domains, which may help to explain why lipid rafts do not form on large scales in vivo.