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Single-particle visualization of viral fusion and its in hibition by an antiviral peptide

UNSPECIFIED (2014) Single-particle visualization of viral fusion and its in hibition by an antiviral peptide. Bachelor's Thesis, Physics.

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The common flu is caused by the influenza virus. The influenza virus is divided in several types: influenza A, influenza B, and influenza C. Each type is divided in different subtypes due to differences in their surface proteins. Here the X31 and the Puerto Rico (PR) viruses are used, both viruses are subtypes of the influenza A type. For any virus to cause illness, it has to deposit its viral genome into the host cell by merging its membrane with the membrane of the host cell and making a pore in the membranes. The virus achieves the merging of the membranes with its surface protein, hemagglutinin. Under the right conditions, hemagglutinin can bind to the membrane of the host cell and pull them together. If the membranes are close enough they can fuse. After fusion a fusion pore will be created so that the viral genome can enter the cell, be replicated, support propagation of the virus, and cause illness. Virus fusion can be visualized by fluorescence microscopy. To achieve this, the viruses will be labeled with fluorescent molecules. After excitation by laser light, they emit light of a red-shifted wavelength which will be imaged with a CCD camera using a so-called Total-Internal-Reflection Microscopy (TIRF) microscope. With the TIRF microscope is a part of a flow channel observed. In this flow channel is a lipid bilayer membrane formed. Viruses will bind to the lipid bilayer and when the pH in the flow channel is lowered the viruses can fuse with the lipid bilayer. When the viruses fuse the intensity of the emitted light will increase. This way virus fusion is visualized. By inhibiting virus fusion, the virus cannot cause illness anymore. The Baker lab at the University of Washington, Seattle (U.S.A.) designed an antiviral peptide to inhibit fusion of the Puerto Rico (PR) virus. To see whether the antiviral peptide indeed inhibits fusion of the PR virus, it will be added to the virus and visualized the same way as without the peptide. If the peptide indeed inhibit fusion, multiple questions will arise. For example: how many of these antiviral peptides are necessary to inhibit fusion? What is the dependence of the number of antiviral peptides to the fusion percentage? To be able to answer these questions the antiviral peptide will be labeled with a fluorescent molecule. This fluorescent molecule is excited with a different laser and emits a different color light than the one used for the virus. From the intensity of one fluorescent molecule on an antiviral peptide and the intensity that arises from the fluorescent molecule(s) of the antiviral peptide(s) that are attached to a virus particle, the number of antiviral peptides on the virus can be determined. With the number of peptides on the virus and the knowledge of whether the virus fused or not, conclusions of the number of peptides necessary for fusion inhibition can be drawn.

Item Type: Thesis (Bachelor's Thesis)
Degree programme: Physics
Thesis type: Bachelor's Thesis
Language: English
Date Deposited: 15 Feb 2018 07:57
Last Modified: 15 Feb 2018 07:57

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