Influenza A virus infections in pigs is a disease of concern to the swine industry and to the ecology and epidemiology of influenza viruses in humans. Pigs have been proposed as a “mixing vessel” for generation of pandemics via reassortment between avian and mammalian viruses. The H1N1pdm 2009 virus probably emerged from swine into humans though reassortment between the recent North American triple reassortant H1N2 swine viruses and Eurasian avian-like swine viruses. Swine influenza viruses of H1N1, H1N2 and H3N2 subtypes have been regularly detected in pigs in most parts of the world. Nevertheless, ecological and virological data on swine influenza is not available in Sri Lanka, and indeed, little documented data is available in the South Asian continent. The swine population in Sri Lanka is about 80,000, and live pigs are not regularly imported to the country. Swine husbandry is largely confined to four neighboring administrative districts in the country. Systematic virological and serological surveillance carried in swine abattoirs in Sri Lanka during 2009-2013 detected H1N1pdm 2009 like virus in local herds. Infection in pigs followed each of the H1N1pdm 2009 outbreaks in humans; October 2009 – January 2010, October 2010 – February 2011 and November 2012 – March 2013, respectively. Genetic, phylogenetic, and epidemiologic analysis of the human, and swine influenza viruses indicated spillover events of H1N1pdm 2009 from humans into pigs, with self-limited transmission and extinction within pig herds. The data also indicated that although H1N1pdm 2009 was able to spill over from humans to swine, it is not ideally adapted to establish sustained transmission among swine in the absence of further reassortment with other swine influenza virus lineages. Theses finding might reflect characteristics of swine husbandry in Sri Lanka, which has a low density pig population and remains isolated from global swine influenza viruses because of the absence of regular cross-border and cross-continental movements of swine. In contrast to some other parts of the world, we failed to isolate established lineages of swine influenza viruses, viz. Classical, North American triple reassortant and European Avian lineages. Sero prevalence to these endemic swine viruses was largely absent in local swine herds. In vitro replicative kinetic study indicated that H1N1pdm 2009 viruses isolated from swine have undergone some adaptation to swine led to decreased fitness for replication in human cells. / published_or_final_version / Public Health / Doctoral / Doctor of Philosophy

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

No description available.

No description available.

Herpetic stromal keratitis (HSK) is an immunopathological and tissue destructive corneal lesion caused by herpes simplex virus (HSV) infection, which induces an intense inflammatory response and finally leads to blindness. Accumulating evidence using the murine model has shown that Th-1 phenotype CD4+ T cells orchestrating the inflammation mainly contribute to the immunopathological reaction in HSV-1 infected cornea. Initially various innate immune cells recruit and produce numerous inflammatory and angiogenic molecules into the corneal stroma those in turn drive the corneal immunopathology. While the basic principles of immunity to the influenza A viruses (IAV) are probably similar for all vertebrates, detailed understanding is based largely on experiments in laboratory mice. Virus clearance is normally mediated via CD8+ effector T cells but, in their absence, the class-switched antibody response can ultimately achieve the same goal. Influenza virus-specific plasma cells and CD8+ T cells persist in the long term and the recall of the CD8+ T cell response can lead to earlier virus clearance. The first part (Part I) of this dissertation focuses on the understanding of HSV-1 induced immunoinflammatory processes in the cornea and the secondary lymphoid tissues and the involvement of immuno-modulatory mechanisms following acute viral infections such as HSV and IAV. The next three parts (Part II-IV) focus on different inflammatory and counter-inflammatory mechanisms that are activated following acute viral infections. Results in Part II evaluate the role of small molecule inhibitors of VEGFR2/src kinase inhibitors in controlling the progression of the inflammatory lesions after ocular HSV infection. Results of the third section show that the host counter inflammatory mechanisms inhibit tissue damage but these may also act to constrain the effectiveness of immunity to acute infections. The fourth section describes the functional significance of HVEM expression on regulatory T cell in their expansion following HSV-1 infection. In this study, experiments were designed to understand the mechanisms involved in the regulation of immunity and resultant immunopathology using HSV-1 and IAV as the model systems and that modulation of these processes can enhance immune response and diminish immunopathology following acute infections.

Genetic immunization, also known as DNA or polynucleotide immunisation, is well documented to induce broad-based immunity in various animal models of infectious and non-infectious diseases. However, the low potency of DNA vaccines has to date precluded the development of commercial vaccines. The aim of this study was to systematically investigate a number of parameters to improve the potency of DNA vaccines for use in chickens, using a low pathogenic avian influenza (LPAI) virus as a proof-of-concept for their ability to produce a humoral immune response. The index virus used in the study was avian influenza virus A/coot/WA/2727/79 (H6N2), isolated from an apparently healthy Eurasian coot in 1979. Prior to any DNA experiments the virus was rigorously characterized. The virus strain was shown to be an H6 subtype by haemaglutination inhibition (HI) testing and as an N2 subtype by gene sequence analysis. The isolate was shown to be able to grow on MDCK cells in the absence of exogenous trypsin. It was further biologically characterized as LPAI with an intravenous pathogenicity index (IVPI) of 0.15 and a motif of 321PQAETRG328 at the cleavage site of the haemagglutinin (HA) protein. It was capable of infecting domestic chickens under experimental conditions with a low level of virus excretion via the cloaca and oropharynx following intravenous or oral and oculonasal inoculation. The full-length HA and nucleoprotein (NP) genes of this H6N2 virus were subsequently cloned into the eukaryotic expression vector VR1012 to generate VR-HA and VR-NP constructs. Six-week-old Hy-Line chickens were intramuscularly injected with either the VR-HA or VR-NP vaccine at different dose rates, with or without lipofectin as adjuvant. Minimal or no detectable antibody was produced, as measured by HI, ELISA and Western blotting-based assay, but high titres of H6-specific HI antibodies appeared 10 days after homologous virus challenge. In contrast to the empty vector controls, there was a significant difference in HI antibody titre between pre- and post-challenge in vaccinated birds, indicating some evidence for the priming effect of the DNA vaccines. Using the frequency of virus shedding as an indicator of protection, lower doses (50 or 100 ¦Ìg per chicken) of either adjuvanted VR-HA or VR-NP vaccine significantly reduced virus shedding in oropharyngeal and cloacal swabs compared to higher doses (300 or 500 ¦Ìg per chicken ) or empty vector control chickens. Although two vaccinations with naked VR-HA alone were not sufficient to induce an effective immune response against a homologous virus challenge, further repeat vaccinations and incorporation of adjuvant did lead to the generation of low to moderate HI antibody titres in some chickens and resulted in no or reduced virus shedding after challenge. Next, to examine the effect of expression vector, three different DNA vectors, pCI, pCI-neo and pVAX1 were used to clone the same HA gene and generate three DNA vaccine constructs. Once again, direct intramuscular injection of the three DNA constructs did not elicit measurable H6-specific HA antibody response in Hy-Line chickens but the 100 µg pCI-HA lipofectin adjuvanted vaccine group showed a significant increase in post-challenge HI titres from the naive control group, indicating that an anamnestic antibody response had been induced by the pCI-HA DNA vaccination. Compared with the controls, the three DNA constructs showed significantly reduced virus shedding in cloacal swabs post virus challenge, suggesting that the three DNA vaccines induced some level of immune response in vaccinated chickens. As with the VR-HA construct, the lower dose groups for each vaccine (50 or 100 g) were more effective at reducing virus shedding from the cloaca than the higher dose group (300 g). To further investigate why the DNA vaccines did not elicit a measurable antibody response, the HA gene incorporating a Kozak enhancer sequence was cloned into an alternative expression vector, pCAGGS, to produce the pCAG-HAk construct. Three-week-old SPF chickens were immunized with this construct either by the intramuscular route (IM) or electroporation (EP). H6 HI antibodies were present in some chickens by 3 weeks after the first IM vaccination and 75% of the chickens vaccinated with 10, 100 or 300 µg pCAG-HAk were antibody positive by 2 weeks after the second IM vaccination. For EP immunization, 87.5% of vaccinated birds seroconverted after the first vaccination and 100% seroconverted after the second vaccination and the H6 HI antibody titres were significantly higher than for chickens vaccinated by IM inoculation. Another group was given a single dose IM vaccination with 100 µg of the pCAG-HAk construct and showed a maximum sero-conversion rate of 53.3% with a peak H6 HI titre of 27 at 5 weeks post-vaccination. This demonstrated that optimization of the expression vector and insertion of a Kozak sequence could synergistically enhance expression of the H6 HA gene and result in a measurable H6 antibody response in SPF chickens. EP was also compared with IM inoculation with the 100 g pCI-HA construct in SPF chickens, resulting in a 50% sero-conversion rate and mean HI titre of 21.3 at 2 weeks after the second vaccination by EP. By comparison, only 25% chickens had trace HI titres by IM inoculation. This indicated that EP was more efficient than IM delivery for both constructs. A codon-optimized complete HA gene from A/coot/WA/2727/79 (H6N2) was then chemically synthesized and cloned into a pCAGGS vector to generate the pCAG-optiHAk construct. SPF chickens immunized twice with either 10 µg or 100 µg of pCAG-optHA showed 37.5% and 87.5% sero-conversion rates respectively, with a mean H6 HI tire of 21.4 and 22.6 at 3 weeks after the second immunization, but the differences were not statistically significant. There were also no significant differences in either the sero-conversion rate or the H6 HI titre between the pCAG-HAk and pCAG-optiHAk groups, suggesting that a codon-optimized HA DNA vaccine did not achieve significantly better immunogenicity than the pCAG-HAk vaccine. In vitro expression of the developed DNA constructs in chicken-, hamster-, monkey- and human-origin cells, as measured by Western blotting and immunofluorescence testing (IFT), showed the strength of H6 HA expression in the following descending order - pCAG-optiHAk/pCAG-HAk, pCI-HAk, VR-HA, pCI-HA, pCIneo-HA and pVAX-HA. The in vivo chicken vaccinations also showed that the pCI-HA construct was more effective than the pCI-neo-HA, and that the pCAG-optiHA or pCAG-HAk constructs were better than pCI-HAk in term of reduction in virus shedding after H6N2 virus challenge. Thus, in vitro HA gene expression directly correlated with the generation of immune responses in vivo, indicating that in vitro studies can be used for pre-selection of expression plasmids prior to development of avian influenza DNA vaccines. Lipofectin as a chemical adjuvant was shown to enhance the DNA-induced immune response but is prohibitively expensive for routine use in poultry vaccines. Thus, an experimental adjuvant for poultry DNA vaccines (Essai) and a new nanoparticle (Phema) adjuvant used for the first time in poultry were compared with conventional aluminum salts (alum) adjuvant in the present study. No HI antibody was detected in any adjuvant-vaccinated Hy-Line chickens following two immunizations. However, in comparison with the naive control group, the alum- and Phema adjuvanted pCAG-HAk groups significantly reduced the frequency of virus shedding in oropharyngeal swabs, but Essai adjuvant was not effective in augmenting the pCAG-HAk vaccine efficacy. This pilot study also emphasised that the traditional aluminum hydroxide adjuvant, either DNA binding or non-binding, may be useful as an adjuvant for enhancing DNA-induced immune responses in chickens owing to its low price and safety record. Overall, DNA immunization with various HA-expressing constructs was shown to be variably effective in inducing immune responses in chickens. The efficacy of DNA vaccines could be synergistically improved by taking appropriate approaches. With continuing research DNA vaccines have the potential to become an important tool for disease prevention and control.

Our innate immunity is our first line of defence against pathogens. We require this immunity to control the numerous viral infections we are challenged with during our lives. However, little is known about the exact molecular mechanisms of our innate immunity, particularly components that have specific antiviral potential. One potent mediator of this antiviral activity is the interferon system. Activation of the interferon system leads to the production of several interferon-induced proteins, which inhibit the multiplication of viruses by distinct mechanisms. A key example of one of these mediators is the human MxA protein. Human MxA has the capacity to inhibit many different viruses from diverse families. In many cases it is proposed that MxA interferes with key viral components, such as incoming or newly formed nucleocapsids. It is speculated that MxA traps and missorts these viral components so they are no longer available for virus production and virus dissemination is inhibited. West Nile virus belongs to Flaviviridae family of viruses and was involved in the outbreak of virus-associated encephalitis in New York City in 1999. In this thesis I show that West Nile virus is insensitive to antiviral activity of MxA and describe how West Nile virus has developed a replication strategy that avoids MxA recognition and activation. I show that virus-induced changes of cytoplasmic membranes provide a protective microenvironment for viral replication and the viral components essential for viral replication. This hypothesis was proven by preventing the formation of these membrane structures with the fungal chemical Brefeldin A. Under these conditions I observed that stable expression of MxA could partially restrict West Nile virus RNA replication. Subsequently, I showed that the assembly mechanism of West Nile virus prevents interaction between the MxA protein and the viral capsid proteins. This was achieved by the use of a trans-packaging cell line whereby the West Nile virus structural proteins are expressed stably in trans instead of in cis from the polyprotein. When this cell line was transfected with a West Nile virus replicon expressing the human MxA protein distinct co-localisation and redistribution of the MxA with West Nile virus capsid proteins into large tubular structures within the cytoplasm of transfected cells was observed. Strikingly, these tubular aggregates are visually analogous to structures observed during infection of MxA expressing cells infected with members of the Bunyaviridae, particularly La Crosse virus. Moreover, retargeting MxA to specific sites of the endoplasmic reticulum in cells transfected with the West Nile virus infectious clone resulted in co-localisation between MxA and the West Nile virus capsid proteins and significantly inhibited the production of infectious particles. These results suggest that the sequestering of viral capsids within cytoplasmic inclusions maybe a conserved mechanism for antiviral activity of the MxA protein across the viruses families and highlight the innate ability of such molecules to recognise key molecular patterns within pathogens. Finally, I sought to exploit the antiviral potential of MxA as a therapeutic agent against infection with Influenza A viruses; viruses that have a very high sensitivity for the antiviral activity of MxA. By expressing MxA from the West Nile virus replicon, infection with the highly pathogenic Influenza virus H5N1 strain could be inhibited in vitro. Furthermore, in vivo studies in Mx-negative mice indicated that intranasal inoculation with MxA expressed from the West Nile virus replicon can protect these mice against an otherwise lethal infection with a low pathogenic Influenza A virus. Taken all together, in this thesis I provide evidence that strongly supports the existence of an evolutionary working mechanism of a significant mediator of our immune system, the antiviral MxA protein. Furthermore, I show how an important human pathogen, such as West Nile virus has evolved a replication strategy to evade this antiviral protein. These results will open new pathways for the development of a new type of antiviral therapies that utilize the potent antiviral activity of the MxA protein.

Our innate immunity is our first line of defence against pathogens. We require this immunity to control the numerous viral infections we are challenged with during our lives. However, little is known about the exact molecular mechanisms of our innate immunity, particularly components that have specific antiviral potential. One potent mediator of this antiviral activity is the interferon system. Activation of the interferon system leads to the production of several interferon-induced proteins, which inhibit the multiplication of viruses by distinct mechanisms. A key example of one of these mediators is the human MxA protein. Human MxA has the capacity to inhibit many different viruses from diverse families. In many cases it is proposed that MxA interferes with key viral components, such as incoming or newly formed nucleocapsids. It is speculated that MxA traps and missorts these viral components so they are no longer available for virus production and virus dissemination is inhibited. West Nile virus belongs to Flaviviridae family of viruses and was involved in the outbreak of virus-associated encephalitis in New York City in 1999. In this thesis I show that West Nile virus is insensitive to antiviral activity of MxA and describe how West Nile virus has developed a replication strategy that avoids MxA recognition and activation. I show that virus-induced changes of cytoplasmic membranes provide a protective microenvironment for viral replication and the viral components essential for viral replication. This hypothesis was proven by preventing the formation of these membrane structures with the fungal chemical Brefeldin A. Under these conditions I observed that stable expression of MxA could partially restrict West Nile virus RNA replication. Subsequently, I showed that the assembly mechanism of West Nile virus prevents interaction between the MxA protein and the viral capsid proteins. This was achieved by the use of a trans-packaging cell line whereby the West Nile virus structural proteins are expressed stably in trans instead of in cis from the polyprotein. When this cell line was transfected with a West Nile virus replicon expressing the human MxA protein distinct co-localisation and redistribution of the MxA with West Nile virus capsid proteins into large tubular structures within the cytoplasm of transfected cells was observed. Strikingly, these tubular aggregates are visually analogous to structures observed during infection of MxA expressing cells infected with members of the Bunyaviridae, particularly La Crosse virus. Moreover, retargeting MxA to specific sites of the endoplasmic reticulum in cells transfected with the West Nile virus infectious clone resulted in co-localisation between MxA and the West Nile virus capsid proteins and significantly inhibited the production of infectious particles. These results suggest that the sequestering of viral capsids within cytoplasmic inclusions maybe a conserved mechanism for antiviral activity of the MxA protein across the viruses families and highlight the innate ability of such molecules to recognise key molecular patterns within pathogens. Finally, I sought to exploit the antiviral potential of MxA as a therapeutic agent against infection with Influenza A viruses; viruses that have a very high sensitivity for the antiviral activity of MxA. By expressing MxA from the West Nile virus replicon, infection with the highly pathogenic Influenza virus H5N1 strain could be inhibited in vitro. Furthermore, in vivo studies in Mx-negative mice indicated that intranasal inoculation with MxA expressed from the West Nile virus replicon can protect these mice against an otherwise lethal infection with a low pathogenic Influenza A virus. Taken all together, in this thesis I provide evidence that strongly supports the existence of an evolutionary working mechanism of a significant mediator of our immune system, the antiviral MxA protein. Furthermore, I show how an important human pathogen, such as West Nile virus has evolved a replication strategy to evade this antiviral protein. These results will open new pathways for the development of a new type of antiviral therapies that utilize the potent antiviral activity of the MxA protein.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 238-267) Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 194-237) Also available in print.