Respiratory syncytial virus (RSV) infection causes ~64 million cases of respiratory disease and 200,000 deaths annually worldwide, yet there is no broadly effective prophylactic or treatment regimen. RSV can produce acute respiratory illness in patients of all ages but strikes the age extremes, infants and the elderly, with highest frequency presumably due to innate immune deficiencies. A higher morbidity and mortality has been reported for the elderly above 65 years of age, which has been attributed to immune senescence. Efforts to generate an effective vaccine have thus far been unsuccessful. The innate immune system provides the first line of defense against viral pathogens with a repertoire of anatomical barriers, phagocytic immune cells, pattern recognition receptors (PRRs) and antiviral cytokines like interferons (IFNs). The precise mechanism of subversion of innate immunity in young and aged is poorly understood. A better understanding of innate immune pathways is expected to aid in the development of appropriate vaccines or prophylactics for these high-risk groups. Previously, the RSV nonstructural protein 1 (NS1) was shown to antagonize IFN responses by disrupting components of the innate immune system, although the mechanism is not well defined. We hypothesized that NS1 targets constituents of the PRR pathways to evade innate immunity and thus ensure viral survival. Using microscopy and co-immunoprecipitation assays, we found that NS1 localizes to the mitochondria and binds to the mitochondrially associated adaptor protein MAVS, thus preventing MAVS interaction with the RNA helicase, RIG-I. Expression of NS1 was also correlated with upstream IFN-response regulator, LGP2, and its expression was inducible in the absence of a viral infection. Tetracycline-inducible expression of recombinant NS1 in a cell model also promoted viral replication and emphasizes the key contribution of NS1 to RSV survival. Through this study, we demonstrated a mechanism for RSV NS1 in the disruption of early innate responses through mitochondrial localization and alteration of the RLH signaling. Whereas the above studies showed the importance RSV-induced innate immune pathways, whether the expression and signaling of innate immune pathways were adversely affected upon RSV infection in the high-risk groups remains unknown. Since elderly individuals are at an increased risk for severe bronchiolitis and RSV-induced pneumonia, often resulting in hospitalization and medical intervention and adaptive immune cell functionality and responsiveness reportedly decline with age, we hypothesized a similar age-related deterioration of the innate antiviral system. In this investigation, we used an aged mouse model to correlate age-associated changes in innate immune gene expression with RSV pathology. Of 84 antiviral genes examined, five genes including RIG-I, IFNAR1, TLR8, IL-1Β, and osteopontin (OPN) were associated with both age and infection. In response to RSV infection, aged mice had delayed induction of antiviral genes and diminished ability to secrete IL-6 in response to TLR7/8 agonist in primary alveolar macrophages. Lungs from aged, RSV-infected mice had increased cellular infiltration and prolonged infection as compared to young mice. In summary, age-related decline in expression and functionality of antiviral defenses were correlated with enhance RSV-induced lung disease in aged mice. In the absence of infection, aged mice chronically overproduced IL-1Β and OPN relative to young mice. Upon infection, aged mice had impaired ability to secrete higher levels of IL-1Β and mucus. In contrast, OPN secretion remained high and prolonged in aged mice throughout infection. The age-related decline in host antiviral gene induction and delayed cytokine production correlated with enhanced disease pathology. Using a transgenic strain of mice deficient in OPN (OPN-KO), we observed greater resistance to RSV and enhanced secretion of mucus, but unaltered cellular infiltration into the lungs. Therefore, OPN overproduction and defective mucus production likely contribute to pathology in aged mice. These findings demonstrate that RSV targets the innate virus recognition and antiviral cytokine activation pathways but also that the antiviral defense system is significantly affected by age. Consequently, efforts to generate vaccines or develop therapies that stimulate IFN induction may prove unsuccessful in the elderly given that RSV virulence factors and age weaken these responses. This study contributes to our understanding of how aging relates to the RSV subversion of the host antiviral response and should help with the development of better antiviral therapies suited to the growing elderly population.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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Ypač svarbią reikšmę Lietuvos žemės ūkiui turi viena iš gyvulininkystės šakų – galvijininkystė. Jos plėtrai didelę žalą daro galvijų užkrečiamosios, ypač virusinės, ligos, todėl būtina greitai, tiksliai ir efektyviai diagnozuoti galvijų virusines ligas, užkirsti kelią jų plitimui Lietuvoje. Kvėpavimo ir virškinimo trakto ligomis galvijų prieauglis serga visose pramoninės gyvulininkystės šalyse, dėl šių ligų galvijai gaišta žymiai dažniau, nei nuo reprodukcijos, medžiagų apykaitos ligų ar mastitų. Didesnį veršelių susirgimų skaičių ir gaišimą nuo enteritų bei respiratorinių ligų galima būtų paaiškinti dideliu virusinių ligų – GVD, IGR, PG-3, RSV, adenovirusų ,RV, CV ir bakterinių infekcijų išplitimu. Mūsų darbo tikslas buvo atlikti galvijų užkrečiamųjų virusinių ligų paplitimo analizę, ištirti CV, RV, RSV ir adenovirusų paplitimą galvijų bandose. Dėl rota ir korona virusų buvo ištirti 56 išmatų mėginiai, dėl antikūnų prieš adenovirusą – 20, RS virusą 28 kraujo serumo mėginiai. Šiems tyrimams buvo naudojami komerciniai standartizuoti imunofermentinės analizės (IFA) rinkiniai. Veršelių 30 kraujo serumo mėginių buvo ištirti pusiau kiekybiniu natrio sulfito precipitacijos metodu, dėl imunoglobulinų kiekio nustatymo. Tyrimai atlikti LSMU VA Užkrečiamųjų ligų katedroje ir Nacionalinio maisto ir veterinarijos rizikos vertinimo instituto Virusologijos skyriuje 2013 metais. Įvertinus padėtį 3 ūkiuose, kur buvo atlikti ligų tyrimai, nustatyta, kad rotavirusine infekcija vidutiniškai... [toliau žr. visą tekstą] / Cattle take a very important place in Lithuanian agriculture. Since the development of livestock can be seriously affected by contagious diseases, especially viral ones, it is highly important quickly, accurately and efficiently diagnose viral disease of cattle, and to prevent its spreading in Lithuania. In all livestock farming industry countries, cattle offspring are suffering from respiratory and digestive tract diseases. They are dying from these diseases more often than from reproductive, metabolic diseases and mastitis. Increasing calves’ cases and mortality rate from enteritis and respiratory diseases can be explained by the high viral diseases such as GVD, IGR, PG-3, RSV, adenovirus, RV, CV and bacterial infections spread. The goal of our study was to analyze the spread of livestock infectious viral diseases and examine the CV, RV, RVS and adenovirus prevalence in the cattle herds. 56 fecal samples were tested for the rotavirus and coronavirus, 20 – for antibodies against adenovirus, and 28 blood serum samples for RS virus. To test these samples, we used standardized commercial enzyme-linked immunosorbent assay (ELISA) kits. 30 blood serum samples of calves were analyzed by quantitative sodium sulfite precipitation method for the determination of immunoglobulin. The tests took place at LSMU VA Department of Infectious Diseases and National Food and Veterinary Risk Assessment Institute, Department of Virology in 2013. Cattle from 3 farms were assessed for anticipated... [to full text]

The CD40 ligand (CD40L) molecule is central to innate and adaptive immunity. CD40L expression is very tightly regulated whereas its CD40 receptor is constitutively expressed by many different cell types. CD40L is expressed transiently on helper T cells (Th) only after activation by specific immune recognition molecules carried by professional antigen presenting cells, in particular, dendritic cells (DC). CD40L subsequently binds to CD40 on DC to enable full Th activation. CD40 ligated DC produce interleukin-12 (IL-12) and contribute both to the development of IFNγ-secreting natural killer cells, a vital component of innate immunity, and of IFNγ-secreting type 1 Th (Th1) cells. CD40 ligated DC also contribute to the development of IL-4- and IL-10-secreting Th2 cells. CD40L on Th cells also binds CD40 on macrophages to enhance their cytotoxic functions. CD40L-expressing Th cells provide the ‘help’ pivotally required to activate other components of adaptive immunity responsible both for clearing invading pathogens and generating the memory cells required to prevent re-infection. Th-supplied CD40L binds (i) B cell CD40 to switch production of antibodies to more potent effector molecules that have higher avidity for antigen, and (ii) DC CD40 to prime then expand antigen-specific cytotoxic T lymphocytes (CTL). Activated NK cells and CTL are required both to eradicate malignant cells and cells infected with viruses or other intracellular pathogens. Genetic CD40L deficiency causes the very rare HyperIgM Syndrome Type 1 (HIGM1), which is realistically modelled by genetically engineered CD40L-deficient mice. Neither CD40L-deficient patients nor mice make effective antibodies or mount cellular immune responses that would defend them against intracellular pathogens such as parasites. Consequently, the only potentially curative therapy is allogeneic stem cell transplantation or CD40L gene replacement. Here, we used a retroviral vector, which constitutively expressed CD40L, to genetically modify CD40L-deficient bone marrow cells, which were used to reconstitute partially the immunity of CD40L-deficient mice. The crucial importance of tight regulation of CD40L expression was revealed when these mice later developed lethal thymic T cell malignancy. Growing tumours escape immune vigilance by genetic alterations that reduce their sensitivity to IFNγ. Using murine tumour models, we incorporated transgenic CD40L expression in therapeutic tumour vaccines to show that CD40L gene transfer augmented the immunogenicity of the host’s tumour thus reducing its tumorigenicity. We translated this finding clinically to safety and immunogenicity testing of a transgenic CD40L- and IL-2- expressing leukaemia vaccine. Finally, the common viral respiratory pathogen, respiratory syncytial virus (RSV) mainly infects young infants and the elderly to cause potentially lethal pneumonia. Both groups have reduced cellular and humoral immunity, which predisposes them to re-infection with RSV. Using a murine model, we showed first that simultaneous adenoviral expression of CD40L augmented primary RSV-specific Th1 responses that were associated with accelerated pulmonary viral clearance. Second, we showed that expression of CD40L in RSV-F and RSV-G subunit DNA vaccines elevated antibody and cellular immune responses to RSV challenge four and eight months after the initial immunisation. These results demonstrate the potent ability of CD40L gene transfer to solve the absolute immune deficiency caused by genetic lesions of CD40L. However, physiological regulation of the transgene is required to prevent serious adverse consequences. In contrast, no adverse effects were observed after transgenic CD40L expression was used to overcome relative immune deficiencies imposed by malignancy and RSV infection. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1298200 / Thesis (Ph.D.) - University of Adelaide, School of Medicine, 2007

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / "May 1997." Typescript. Vita. Includes bibliographical references (leaves 210-233). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves: 210-233). Also available on the Internet.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 108-121). Also available in print.

Thèse (Ph. D.)--Université Laval, 2009. / Bibliogr.: f. 107-115. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thesis (M.Med.Sc.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 42-47). Also available in print.