No description available.

No description available.

Proper mRNA nuclear export is essential for harmonious growth and maintenance of a cell. An effective weapon influenza virus employs to hijack a host cell is its ability to inhibit such export. Exactly how influenza virus achieves this inhibition is not fully known. Here, we demonstrate that upon infection, influenza virus degrades two nucleopore proteins (Nup98 and Nup96), which play a key role in mRNA nuclear export. Also, a main virulence factor of influenza virus (non-structural protein 1, NS1) binds directly to NXF1 and E1B-AP5, two key constituents of the mRNA export pathway (NXF1/NXT pathway) responsible for exporting bulk (~70%) mRNA from the nucleus. By increasing the expression levels of members of the NXF1/NXT pathway, we were able to reverse NS1-mediated inhibition of gene expression. On the other hand, by decreasing the levels of members of the NXF1/NXT pathway, we demonstrated that host cells become more sensitive to influenza virus infection and produce more viral particles. These results demonstrate undiscovered influenza-mediated host interactions that may be used to medicinally inhibit influenza virus. To this end, high-throughput screens were designed to identify small molecule antagonists of both NS1-mediated inhibition of gene expression and influenza virus-mediated cell death. Seventy-one compounds were identified, and the most potent molecule (named compound #8) was examined further. We found that compound #8 releases influenza virus-mediated mRNA nuclear export blockage and decreased viral replication and viral gene expression. Thus, the bulk mRNA nuclear export machinery is vital to antiviral response, and compound #8 enhances its ability to fight the cytopathic effects of NS1 and influenza virus. In conclusion, our data demonstrate that the mRNA export machinery is disrupted by influenza virus, and that this machinery also facilitates an antiviral function. We have also shown that these two events can be manipulated chemically to attenuate the negative effect of the virus and enhance the positive antiviral effect of the mRNA export machinery, thereby providing a powerful, new strategy against the ever-present, global threat of influenza virus.

Influenza A is one of the most prevalent and significant viral infections worldwide, resulting in annual epidemics and occasional pandemics. Upon infection, antiviral drugs targeting the neuraminidase protein and M2 protein are the only treatment options available. However, the emergence of antiviral drug resistance is concerning, therefore the aim of this work was to identify inhibitor molecules that may bind to highly conserved regions of selected internal influenza A proteins. Sequences of the non-structural protein 1 (NS1), nuclear export protein (NEP) and polymerase basic protein 2 (PB2) from all hosts and subtypes were aligned and the degree of amino acid conservation was calculated based on Valdar's scoring method. Missing parts of the experimental structures were predicted using the I-TASSER server and ligand binding hot spots were identified with computational solvent mapping. Selected binding sites in conserved regions were subjected to virtual screening against two compound libraries using AutoDock Vina and AutoDock 4. Two out of twelve top hit compounds predicted to target the NS1 protein showed capability of reducing influenza A H1N1 replication in plaque reduction assays at concentrations below 100 μM, although the target protein and mechanism of action could not be confirmed. For the NEP, conservation analysis was based on 3000 sequences and binding hot spots were located in common areas amongst three structures. Docking results revealed predicted binding affinities of up to -8.95 kcal/mol, and conserved amino acid residues interacting with top compounds include Arg42, Asp43, Lys39, Ile80, Gln101, Leu105, and Val109. For the PB2 protein, conservation analysis was based on ~12,000 sequences and fifteen potential binding hot spots were identified. Docking results revealed predicted binding affinities of up to -10.3 kcal/mol, with top molecules interacting with the highly conserved residues Gln138, Gly222, Ile539, Asn540, Gly541, Tyr531 and Thr530. The findings from this research could provide starting points for in vitro experiments, as well as the development of antiviral drugs that function to inhibit influenza A replication without leading to resistance.

Influenza is a seasonal acute respiratory infection, causing millions of illnesses worldwide on a yearly basis. A common subtype, the influenza A virus (IAV), is a single stranded RNA virus, that similarly to other subtypes, targets epithelial cells. The best way to protect against the virus is through vaccination. Vaccine induced protection is mediated through the generation of adaptive CD4 and CD8 T cells, as well as antibody producing B Cells. Although generally thought of as helper cells, previous research has highlighted additional roles of memory CD4 T cells in mediating protection against IAV beyond their helper function. More specifically they have been shown to enhance innate inflammatory responses and facilitate the recruitment of innate cell populations; including a recently discovered population of Innate Lymphoid Cells (ILC). Previous research has shown ILCs to have a key role in tissue repair and limiting tissue damage following infection. Whether memory cell response, during protective recall, modulates ILC repair function is currently not well understood. To better understand the possible regulation of ILCs by memory cells, we utilized a molecular technique called reverse transcription polymerase chain reaction (RT-qPCR), to assess select innate lymphoid cell associated protein expression following IAV challenge and rechallenge. We hypothesize, that memory cells drive differential expression of ILC associated repair proteins to assist in a faster and more efficient mobilization of repair processes following pathogenic invasion. Our goal is to highlight and better understand the regulatory and inflammatory responses memory cells provide following viral infection, as these may lead to key steps in the development of long lasting and efficacious vaccines.

No description available.

A group of Influenza viruses, RNA containing viruses of the Orthomyxoviridae family, consists of Influenza virus types A-D and has been known to cause the Flu, a respiratory illness associated with numerous detrimental symptoms that can lead to death. Influenza A virus (IAV) is constantly changing and is capable of causing pandemics. Currently used diagnostic methods include virus culturing, immunoassays including rapid influenza detection tests (RIDTs), and molecular assays including those based on RT-PCR. Most of the methods can be only performed in the certified diagnostic laboratories equipped with sophisticated instrumentation and/or special biosafety facilities. The results using these methods are not available on a timely basis. RIDTs provide response within 15 minutes but are unable to differentiate between the IAV subtypes. New diagnostic technique, which allows reliable detection of the influenza virus infection and virus genotyping at point-of-care setting, are needed to prevent the spread of the virus and the occurrence of a pandemic. In this project, we propose to use split G-quadruplex (G4) peroxidase probes targeting a fragment of the IAV genome amplified using an isothermal RNA amplification reaction for the detection of IAV infection and virus genotyping. The probes selectively report the virus RNA target with a color change, which can be read by the naked eye. They are capable of differentiating the targets containing as little as a single-nucleotide variation in their sequences. This study aims to optimize the probes, test their selectivity, and calculate the detection limit.

Sialic acids (Sias) are receptors for influenza A viruses (IAVs). The influence of individual sialyltransferases on the Sias expressed is not understood. Also, sensitive methods to distinguish Sias on the cells are lacking. Our goals were to establish a method to detect Sias on the cell and to evaluate the importance of CMP-sialic acid transporter (SLC35A1) and ST6 beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1) for IAV infection. Aproximity ligation assay was established to detect and quantify Sias.

In this study, the expression of HA, a key immunogenic protein of influenza viruses, in insect cells was compared using three baculovirus expression strategies: protein over-expression, surface (GP64) display, and capsid (VP39) display. Further, a recombinant virus expressing NA, another immunogenic influenza virus protein, was generated and fused to an HA epitope-tag. Western immunoblot using various antibodies, including those against HA, demonstrated the expression of HA and NA for all recombinant viruses. HA showed stronger expression when fused to the C-terminus of VP39 than the N-terminus, but unlike other expression methods, there was no observable cleavage of HA in VP39-displayed viruses. Cells infected with only over-expressed and surfaced-displayed HA were biologically active, and capable of hemadsorption and hemagglutination of chicken red blood cells. These results suggest that GP64 display or over-expression are the most efficacious modes of HA-expression for use as antigen to detect anti-HA antibodies in poultry. / NSERC, OGS, OMAFRA, CPRC

Wild birds are the natural reservoirs of Influenza A viruses (IAVs) which cause occasional pandemics and seasonal epidemics. Avian IAVs can be transmitted from wild birds to domestic poultry, low mammals, and humans. It is well accepted that avian IAVs prefer to sialic acids (Sia) α2,3-linked galactose (SAα2,3-Gal), whereas human IAVs to α2,6-linked galactose (SAα2,6-Gal). However, SAα2,3-Gal in wild bird tissues is widely distributed with little variation while some subtypes of avian IAVs have species preference. The different isolation rates among wild bird species cannot be explained by avian IAVs binding to SAα2,3-Gal alone. Specifically, this dissertation had the following aims: Firstly, to determine distribution of glycan receptors across respiratory and gastrointestinal tissues of wild birds and domestic poultry; Secondly, to determine the viral-receptor binding specificity of avian IAVs; Thirdly, to understand the role of glycan motifs in shaping virus evolution during the natural history of IAVs, especially from wild bird to poultry transmission. We found that avian H7 and H10 viruses acquired the binding ability to SAα2,6-Gal without adaptation, furthermore, we evaluated one of these H10 virus that possess the ability of binding to SAα2,6-Gal in ferret model and found it could cause aerosol and contact transmissions. On the other hand, H7 viruses have strong binding avidity to SLex which are present widely in epithelial cells of chicken trachea, which could facilitate the transmission of avian H7 viruses from waterfowl to poultry. Lastly, we found that H7 viruses from waterfowl bound both Neu5Ac and Neu5Gc while chicken isolates from China only prefer to Neu5Ac. Of interest, we found Neu5Gc was found in mallards but not in chickens, which indicated that viruses reduce or lost binding ability to Neu5Gc by adapting in chicken. In summary, this dissertation focused on certain subtypes of avian IAVs, which have caused threats to domestic poultry and public health, and primary avian species for influenza risk. The glycan substructures may play an important role in avian IAVs transmission and adaptation. The knowledge derived from this dissertation will help identify species for influenza surveillance in wild birds and facilitate risk assessment of avian IAVs.