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Respiratory MedicineResearch within the Respiratory Medicine Research Group is focused on two viruses which together account for majority of the respiratory viral infections in the world, rhinovirus and respiratory syncytial virus. Rhinovirus is the single most common cause of upper respiratory tract disease and respiratory syncytial virus is the most common cause of lower respiratory tract disease. Both viruses infect throughout life; severe disease is observed in infants and the elderly. Infection with both viruses has been implicated in asthma attacks; there is also evidence that infection with respiratory syncytial virus in infancy may also lead to future development of asthma. Asthma is increasingly common and affects up to 30% of the population in westernised countries; 600,000 people in Victoria, 2 million in Australia and 150 million people worldwide have asthma. Recent studies have suggested that asthma may cause as many as 4 deaths per week in Victoria, and 16 deaths per week nationwide. World-wide, the economic costs associated with asthma are estimated to exceed those of TB and HIV/AIDS combined. Rhinovirus ResearchAsthma attacks are implicated in the morbidity and mortality associated with asthma and 70% of these attacks are caused by respiratory tract infection by rhinovirus. There is no vaccine or effective antiviral treatment for rhinovirus infection because there are more than 100 serotypes of rhinovirus, making vaccine development difficult. In view of the high cost associated with asthma attacks and the increasing mortality rate, there is an urgent need for new methods of treating infection and minimizing the severity of asthma attacks. Asthma is a chronic inflammatory disease of the lungs, and symptoms of asthma attacks are caused by the host’s inflammatory response to the virus, not by the virus itself. It is important to understand exactly how the virus affects the inflammatory response in order to manage asthma attacks. Research in our group is directed towards understanding the mechanisms used by rhinovirus to induce adverse inflammatory responses in lung cells. Our group has shown that rhinovirus induces interleukins and chemokines (for example, IL-8 and ENA-78) in primary lung epithelial cells [1]. IL-8 and ENA-78 are potent chemotactic factors for neutrophils and are known to play a role in asthma. There is an also an eosinophil influx observed in allergic asthma and in rhinovirus exacerbations. Eotaxin is the major chemokine held responsible for this influx and we have also shown that rhinovirus infection induces eotaxin in primary epithelial cells. The extreme inflammatory responses observed in asthma attacks cannot be explained by the limited damage observed in the epithelial cell layer. We believe that the epithelial infection by rhinovirus spreads to the subepithelial layer, leading to amplified inflammatory responses. Currently we are investigating the effect of rhinovirus infection on induction of chemotactic and angiogenic factors in the subepithelial fibroblast layers of the airway wall. Respiratory Syncytial Virus Research (in collaboration with Professor David Jans, Department of Biochemistry and Molecular Biology, Monash University)Respiratory syncytial virus (RSV) is the major cause of viral pneumonia in infants and the elderly worldwide; by the age of 3 years everyone has had at least one episode of RSV infection. In premature babies, in babies with congenital heart or lung disease, and in immunocompromised adults, RSV infection invariably leads to severe disease requiring hospitalization and may be fatal. In any one year, about 90,000 children will be hospitalised due to RSV and 4,500 will die. In Australia, about 100,000 infants are infected by RSV every winter. The estimated annual cost of this disease in Victoria is between $1 and $4 million. Mechanisms underlying the pathogenesis induced by RSV are mostly unknown. RSV induced bronchiolitis in infancy may predispose to asthma later in life. The major structural protein of the RSV particle is the matrix (M) protein. We have investigated the movement pathways of the M protein in RSV infected cells and found that M is present in the cell nucleus and can inhibit cell transcription [2]. This inhibition of the cellular synthetic machinery may cause the observed pathogenesis in RSV infections. We do not know how M is transported across the nuclear membrane, or how it inhibits cellular transcription. Current research is focussed on elucidating these mechanisms. Following are some of the current Honours projects on offer within our group. Supervisor: Associate Professor Phil Bardin Project 1 Aim: To clarify the mechanisms whereby rhinovirus induces chemokine responses. Infection of lung fibroblasts and epithelial cells by rhinovirus (RV) leads to increased transcription and secretion of various chemokines and angiogenic factors [1]. Chemokine release in response to RV may be induced at various stages of virus infection. Binding of RV to receptor, presence of the viral dsRNA, viral protein expression and virion assembly may all singly or together, contribute to cellular responses. Using chemical agents to interfere with each of these processes, we will investigate exactly which step in the infection cycle is essential for the observed chemokine response. Techniques A number of different techniques are being used in this project, including tissue culture and virology techniques, confocal microscopy, ELISA, real-time PCR, subcellular fractionation, western blots and electrophoretic mobility shift assays. Project 2Aim: Effect of rhinovirus infection on production of macrophage migration inhibitory factor (MIF) (in collaboration with Associate Professor Eric Morand, Department of Medicine(MMC)) Glucocorticoids (GC) are the major line of treatment for management of asthma and asthma attacks. Unfortunately, GCs have only a partial affect on RV induced disease. This observation suggests that RV infection induces the production of a GC antagonist and MIF has been shown to antagonize the affects of GCs; in pilot experiments we have found large quantities of MIF secreted by cells on RV infection. In this project induction of MIF transcription and secretion by RV infection will be analyzed. Techniques A number of techniques will be used in this project, including primary cell culture, virological techniques, ELISA, real-time PCR and confocal microscopy. Project 3Aim: Secondary effects of rhinovirus infection on lung cells IL-8 production is elevated in the airways of people infected with rhinovirus and it plays a major role to induce the tissue neutrophilia that is observed. RV infection of cells leads to an increase in IL-8 transcription and secretion. However, RV infection of cells leads to inhibition of cellular transcription. These two disparate sets of data suggest that RV infection leads to production of certain factors that affect the surrounding uninfected cells inducing IL-8 production. In studies culture supernatants from RV infected cells will be transferred to uninfected cells and affects on IL-8 studied by ELISA and RT-PCR. We will also determine nuclear translocation of transcription factors that have been implicated in the induction of IL-8; both in RV infected and uninfected cells. Techniques A number of different techniques will be used in this project, including tissue culture and virology techniques, confocal microscopy, ELISA, real-time PCR, subcellular fractionation, western blots and electrophoretic mobility shift assays. Project 4Aim: To determine the mechanism of nuclear import of RSV M protein (in collaboration with Professor David Jans, Department of Biochemstry and Molecular Biology) Nuclear transport of proteins is an active process and is regulated by the nuclear pore complex, a complex of more than 50 proteins [3]. Recently we have found that M protein can interact with importin β, a major component of the nuclear pore complex. We aim to further define the mechanism of nuclear import of M protein. In vitro transport assays will be performed with M protein in the presence of specific antibodies to importin β. These experiments will help to determine if binding to importin β is sufficient for nuclear import of M protein. Using truncated and mutated recombinant M proteins, we will determine the domain of M required for nuclear import. Techniques: A range of techniques will be used in this project, including plasmid construction and deletion analysis, bacterial expression, protein purification, protein interactions by ELISA and mobility shift assays, and in vitro transport assays. Project 5Aim: To determine how RSV M protein inhibits cellular transcription (in collaboration with Professor David Jans, Department of Biochemstry and Molecular Biology) Recent work in our laboratory suggests that M protein may inhibit transcription by binding to the DNA template and so hindering the ‘docking’ of the transcription complex. We will confirm if this is so by electrophoretic mobility shift assays (EMSA) using M protein. We will then use various truncated forms of the M protein in EMSA to determine the domain within M protein responsible for binding to DNA. The mutant deficient in DNA binding will be used in in vitro transcription assays to finally confirm that DNA binding is indeed the mechanism of transcription inhibition by M protein. Techniques: A range of techniques will be used, including plasmid construction and deletion analysis, bacterial expression, protein purification, EMSA, in vitro transcription assays and subcellular fractionation. References 1. Donninger H, Glashoff R, Haitchi H-M, et al. Rhinovirus induction of the CXC chemokine ENA-78 in bronchial epithelium. Journal of Infectious Diseases 2003;187:1809-1817 |