Influenza A virus (IAV) is one of the most ubiquitous respiratory viruses in the world, causing significant disease burden in the United States and abroad. Current vaccination strategies that target the generation of humoral immunity offer limited heterosubtypic protection; T cells offer cross-strain protection and the promise of universal immunity against IAV. Local tissue immunity plays a key role in pathogen clearance and tissue protection, particularly in the form of tissue resident memory T cells (TRM), which are a non-circulating memory T cell subset that have been shown in a variety of tissue sites to be superior mediators of protection compared to circulating memory T cells. At the same time, T cell immunity has been associated with inflammatory processes that may also lead to lung immunopathology. How lung tissue localized T cell immunity mediates its protection during a recall response to IAV challenge is not well understood. Using the lymphocyte sequestering drug FTY720, we show that primary infection with H3N2 IAV strain X31 provides tissue localized heterosubtypic immunity independently of humoral immunity against an H1N1 PR8 IAV strain. Within the lung resident niche, the recall response drives faster CD4+ and CD8+ T cell expansion compared to a primary infection. This rapid T cell expansion resulted from in situ TRM proliferation that was augmented by the migration of peripheral T cells. By tracking a naïve T cell population specific for the IAV strain used in secondary challenge, we demonstrate that influenza-specific T cells, including those specific for newly introduced antigens, migrate to the lung niche from the local mediastinal lymph node (medLN) where both CD4+ and CD8+ T cells experience enhanced priming and proliferation. We further show that primary infection fortifies the medLN with persistently increased numbers of T cells as well as both CD103+ and CD103- conventional dendritic cells (cDCs) that are transcriptionally similar to cDCs in an infection naïve mouse. By depleting Zbtb46+ cDCs, we determine that cDC fortification is a crucial mechanism for enhanced T cell priming and expansion in the medLN during a recall response. We also found that lung localized CD4+ T cell responses exhibit significant immunomodulatory function. Polyclonal lung CD4+ TRM generated by influenza infection as well as lung OT-II TRM exhibit increased production of antiviral inflammatory cytokines in addition to enhanced IL-10 family cytokine production compared to splenic CD4+ effector memory T cells (TEM). During a heterosubtypic challenge, we further observed that lung niche non-TRM CD4+ T cells produce significantly more in situ IL-10 compared to a primary infection, which modulated airway IFN-ɣ and TNF-α production without any depreciation in viral clearance. Immunomodulatory characteristics of a recall response was reflected in lung tissue-wide transcriptional downregulation of innate responses such as type I IFN responses compared to a primary infection. This work demonstrates the dual antiviral and immunomodulatory protective role of enhanced tissue-localized T cell responses during the recall response to IAV challenge.

No description available.

Emergence of avian origin and equine origin canine influenza viruses (CIVs) in Asia and the United States brings important concerns. Humans are in closer and more frequent contact with dogs than other common hosts of influenza. Thus, CIV is a potential threat to human health. However, little is known about the determinants of CIV host tropism or the transmissibility of CIVs to humans. An amino acid change (W222L) was implicated in modifying hemagglutinin receptor binding by CIV. This was tested using reverse genetics, glycan microarray and virus histochemistry. Glycan microarray demonstrated that avian-origin CIV (H3N2-222W) bind predominantly to alpha-2, 3 linked glycans. Virus histochemistry indicated that rH3N2-222L had higher binding affinity with epithelial cilia of canine tracheal tissue and weaker binding with avian tracheal tissue. Ferret infection demonstrated that the avian-origin H3N2 CIV could cause infection and limited to rhinitis, suggesting that CIV could infect humans.

No description available.

No description available.

Despite formidable advances in virology and medicine in recent decades, we know remarkably little about the dynamics of the influenza virus in the environment during transmission between hosts. There is still controversy over the relative importance of various transmission routes, and the seasonality of influenza remains unexplained. This work focuses on developing new knowledge about influenza transmission via the airborne route and the virus' inter-host dynamics in droplets and aerosols. We measured airborne concentrations of influenza A viruses (IAVs) and size distributions of their carrier aerosols in a health center, a daycare center, and airplanes. Results indicate that the majority of viruses are associated with aerosols smaller than 2.5 µm and that concentrations are sufficient to induce infection. We further modeled the fate and transport of IAV-laden droplets expelled from a cough into a room, as a function of relative humidity (RH) and droplet size. The model shows that airborne concentrations of infectious IAV vary with RH through its influence on virus inactivation and droplet size, which shrinks due to evaporation. IAVs associated with large droplets are removed mostly by settling, while those associated with aerosols smaller than 5 µm are removed mainly by ventilation and inactivation. To investigate the relationship between RH and influenza transmission further, we measured the viability of IAV in droplets at varying RHs. Results suggest that there exist three regimes defined by RH: physiological conditions (~100% RH) with high viability, concentrated conditions (~50% to ~99% RH) with lower viability, and dry conditions (<~50% RH) with high viability. A droplet's extent of evaporation, which is determined by RH, affects solute concentrations in the droplet, and these appear to influence viability. This research considerably advances the current understanding of the dynamics of the influenza virus while it is airborne and provides an explanation for influenza's seasonality. Increased influenza activity in winter in temperate regions could be due to greater potential for IAV carrier aerosols to remain airborne and higher viability of IAV at low RH. In tropical regions, transmission could be enhanced due to better survival of IAV at extremely high RH. / Ph. D.

A 4.0 Kb (2.64 Mdal) plasmid was isolated from a fowl cholera strain of Pasteurella multocida (the Larsen strain) by alkaline lysis and cesium chloride purification. The plasmid, designated pLAR-1, was characterized in terms of its size and restriction sites. The restriction patterns produced by fourteen endonucleases were used to generate a restriction map. Five restriction enzymes cleaved the plasmid at multiple sites. Two enzymes, Bgl II and Sal I had unique sites on pLAR-1. Twelve of the fifty six strains of P. multocida surveyed contained plasmids of different sizes which hybridized to pLAR-1. Strains containing homologous plasmids were variable in serotype, dermonecrotoxin production, and origin (both in terms of the host and locale). pLAR-1 did not encode any of the enzymes necessary for the biochemical pathways contained within the API-20E strip or siderophore production. pLAR-1 was cloned into the BamH I site of pBR322. Resultant clones were approximately 8.363 Kb in length, ampicillin resistant and tetracycline sensitive. The pLAR-1 / Master of Science

The poultry-to-human transmission of the influenza virus and the recent H1Nl influenza pandemic have become major concerns worldwide. The nucleoprotein (NP) of influenza virus binds the RNA genome and plays essential role in transcription and replication during the virus life cycle. / The study leads to a better understanding towards the RNP organization of influenza virus and provides information for the future design of anti-influenza agents. / We have also shown, by RNP reconstitution assay and co-immunoprecipitation, that the interaction between NP and PB2 is crucial for the proper functioning of the RNP. The functional association of NP and PB2 requires either the PB2 host-determining residue lysine-627 or arginine-630 with the latter involving NP arginine-150 also. Using SPR, we have demonstrated that both residues take part in the direct protein-protein interaction, without the involvement of RNA. These results suggest a dual interaction mechanism between NP and PB2. This may confer replication advantages to the virus, as either one can give an active RNP and explains the increased virulence of avian influenza viruses carrying the E627K mutation in mammalian cells. In addition, our findings identify the NP-PB2 interacting surface, with the PB2 627/630 region facing the RNA binding groove of NP. / We have determined the 3.3 A crystal structure of H5N1 NP, which is composed of head and body domains and a tail loop. Using surface plasmon resonance (SPR), we found the basic loop (residues 73-91) and arginine-rich groove, but mostly a protruding element centering at R174 and R175, to be important in RNA binding. Ribonucleoprotein (RNP) reconstitution assay with these multiple-point and deletion mutants indicate their functional importance towards the transcription-replication activities of the virus polymerase. Single-point mutations at these concerned regions do not have a significant effect on their RNP activities, suggesting that NP mediates RNA-binding through multiple residues. / Ng, Ka Leung. / Adviser: Pang Chui Shaw. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 121-136). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Chan, Wai Hon. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 134-141). / Abstracts in English and Chinese. / Acknowledgements --- p.2 / Abstract --- p.3 / 摘要 --- p.5 / Content --- p.6 / List of Abbreviations and symbols --- p.10 / Chapter Chapter 1 --- Introduction --- p.13 / Chapter 1.1 --- The Severity of Influenza A Virus --- p.14 / Chapter 1.2 --- Introduction to Influenza A Virus --- p.15 / Chapter 1.3 --- What is Nucleoprotein? --- p.17 / Chapter 1.4 --- Multifunctional role of Nucleoprotein --- p.19 / Chapter 1.4.1 --- Interaction of Nucleoprotein with Other Viral Components --- p.19 / Chapter 1.4.1 --- Interaction of Nucleoprotein with Cellular Components --- p.22 / Chapter 1.5 --- Aims of study --- p.23 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Chemical reagents --- p.25 / Chapter 2.1.2 --- Buffers --- p.28 / Chapter 2.1.2.1 --- Preparation of Buffers --- p.28 / Chapter 2.1.2.2 --- Buffer for Common Use --- p.28 / Chapter 2.1.3 --- Plasmids and Strains --- p.31 / Chapter 2.2 --- Methods --- p.32 / Chapter 2.2.1 --- Molecular Cloning --- p.32 / Chapter 2.2.2 --- "Expression of the Recombinant NP-WT/ mutants and importin α1, 3and 5 in E.coli" --- p.46 / Chapter 2.2.3 --- Purification of the NP WT/ variants --- p.50 / Chapter 2.2.4 --- "Purification of importin αl, 3 and 5" --- p.53 / Chapter 2.2.5 --- In vitro interaction study between NP and importin α --- p.56 / Chapter 2.2.6 --- In vivo interaction study between NP and importin α --- p.59 / Chapter 2.2.7 --- In vivo analysis to study NP-NP homo-oligomerization --- p.64 / Chapter 2.2.8 --- In vitro static light scattering analysis to determine NP oligomeric state --- p.68 / Chapter 2.2.9 --- Control experiments to verify NP-polymerases and NP-RNA interaction --- p.69 / Chapter Chapter 3 --- Functional analysis of influenza H5N1 nucleoprotein tail loop for oligomerization and ribonucleoprotein activities / Chapter 3.1 --- Introduction --- p.72 / Chapter 3.2 --- Results --- p.75 / Chapter 3.2.1 --- Tail loop insertion is maintained by intra- and inter-molecular interactions --- p.75 / Chapter 3.2.2 --- NP mutants display defective transcription-replication activity --- p.77 / Chapter 3.2.3 --- Expression and purification of defective NP variants --- p.80 / Chapter 3.2.4 --- Defective NP variants interact with RNA and the polymerase complex --- p.85 / Chapter 3.2.5 --- Defective NP variants possess abnormal oligomeric states in vitro --- p.91 / Chapter 3.2.6 --- NP variants with impaired RNP activity cannot form homo-oligomers in vivo --- p.95 / Chapter 3.3 --- Discussion --- p.98 / Chapter Chapter 4 --- Biophysical characterization of the interaction between influenza nucleoprotein and importin α / Chapter 4.1 --- Introduction --- p.105 / Chapter 4.2 --- Results --- p.108 / Chapter 4.2.1 --- Expression and Purification of NP-WT/ NLS variants and Importin a --- p.108 / Chapter 4.2.2 --- Pull down Assay --- p.113 / Chapter 4.2.3 --- Light scattering analysis (NP-lmportin α5) --- p.114 / Chapter 4.2.4 --- Binding assay of NP NLS variants with importin α5 --- p.115 / Chapter 4.2.5 --- BIAcore 3000 Surface Plasmon Resonance (NP-lmportin α5) --- p.118 / Chapter 4.2.6 --- QRT-PCR (NP-lmportin a5) --- p.123 / Chapter 4.3 --- Discussion --- p.125 / References --- p.134

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.