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postgraduate thesis: Effect of overexpression of B4GalNT2 in MDCK on influenza viral infection
Title | Effect of overexpression of B4GalNT2 in MDCK on influenza viral infection |
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Authors | |
Advisors | |
Issue Date | 2018 |
Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
Citation | Wong, H. [黃可謙]. (2018). Effect of overexpression of B4GalNT2 in MDCK on influenza viral infection. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. |
Abstract | Influenza viruses caused pandemics in the last century. Multiple avian viruses have been found to have infected human successfully. One of the hallmarks for influenza A virus to adapt human respiratory tract is the change in receptor preference from avian α2,3- to human α2,6-linked sialic acid receptors. Thus, it is essential to deepen our knowledge on virus-receptor interaction. Previously therespiratory tract of ferret was found to contain a high density of Sda antigens and could potentially be resistant to avian influenza viral binding and infection. A MDCK cell line overexpressing human B4GalNT2 gene was established by lentiviral transduction. B4GalNT2 protein expression was confirmed with Western blot. Glycosylation profile of the transduced cells was compared to that of wild type cell using immunocytochemistry and enzyme-linked-lectin assay. Influenza viruses were selected according to their receptor binding preferences previously determined by glycan array. Single and multiple cycle growth kinetics were determined using plaque assay. Nucleoprotein positive cells were stained with fluorophore conjugated antibody and acquired by FACS to study the proportions of infected cells. The proportions of cells infected by H5 HA pseudo-particles with GFP reporter was also determined by FACS. Viral attachments were compared through the mean fluorescent intensity of cells incubated with fluorophore labeled virus.
B4GalNT2 protein was confirmed to be expressed in the transduced cells, MDCK-B4GalNT2. MDCK-B4GalNT2 bound significantly weaker by MAA1 and MAA2, without a detectable change in SNA binding. Wild type MDCK showed negative staining with DBA and CT-1 antibody, but MDCK-B4GalNT2 were stained strongly positive. This result suggested that transduction of B4GalNT2 converted surface α2,3-sialic acid receptors to Sda-like antigens
without affecting the content ofα2,6-sialic acid receptors.
MDCK-B4GalNT2 were infected with PR8 and its PB2 mutants. Viral titer was found to be dropped in MDCK-B4GalNT2 with minimal interference in viral polymerase activity. A panel of influenza viruses selected was used to infect MDCK-B4GalNT2. Viral titers and proportion of infected cells by some strains of virus were depleted in MDCK-B4GalNT2 cells in single cycle growth kinetic assay. The differences in viral titers were less significant in multiple cycle growth. MDCK-B4GalNT2 were also infected less efficiently by H5 HA pseudo-particles. Despite the inhibition effect in MDCK-B4GalNT2 to some strains of virus, there were no correlations with the viral receptor binding preferences. For strains that were inhibited, viral attachments were depleted in MDCK-B4GalNT2. The contrary was found to be true for viruses not inhibited in MDCK-B4GalNT2. As a conclusion, overexpression of B4GalNT2 gene in MDCK cells inhibited infection of some strains of influenza virus during viral attachment.
MDCK-B4GalNT2 can act as a novel cell model with reduced surface α2,3-sialic acid receptor expression. Influenza viral infection was shown to be interfered by alteration of glycosyltransferases expressions. The results suggested that receptor binding preferences of the same virus in glycan array and on cell surface may differ.
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Degree | Master of Philosophy |
Subject | Cell lines Influenza viruses |
Dept/Program | Pathology |
Persistent Identifier | http://hdl.handle.net/10722/265378 |
DC Field | Value | Language |
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dc.contributor.advisor | Nicholls, JM | - |
dc.contributor.advisor | Poon, LML | - |
dc.contributor.author | Wong, Ho-him | - |
dc.contributor.author | 黃可謙 | - |
dc.date.accessioned | 2018-11-29T06:22:30Z | - |
dc.date.available | 2018-11-29T06:22:30Z | - |
dc.date.issued | 2018 | - |
dc.identifier.citation | Wong, H. [黃可謙]. (2018). Effect of overexpression of B4GalNT2 in MDCK on influenza viral infection. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. | - |
dc.identifier.uri | http://hdl.handle.net/10722/265378 | - |
dc.description.abstract | Influenza viruses caused pandemics in the last century. Multiple avian viruses have been found to have infected human successfully. One of the hallmarks for influenza A virus to adapt human respiratory tract is the change in receptor preference from avian α2,3- to human α2,6-linked sialic acid receptors. Thus, it is essential to deepen our knowledge on virus-receptor interaction. Previously therespiratory tract of ferret was found to contain a high density of Sda antigens and could potentially be resistant to avian influenza viral binding and infection. A MDCK cell line overexpressing human B4GalNT2 gene was established by lentiviral transduction. B4GalNT2 protein expression was confirmed with Western blot. Glycosylation profile of the transduced cells was compared to that of wild type cell using immunocytochemistry and enzyme-linked-lectin assay. Influenza viruses were selected according to their receptor binding preferences previously determined by glycan array. Single and multiple cycle growth kinetics were determined using plaque assay. Nucleoprotein positive cells were stained with fluorophore conjugated antibody and acquired by FACS to study the proportions of infected cells. The proportions of cells infected by H5 HA pseudo-particles with GFP reporter was also determined by FACS. Viral attachments were compared through the mean fluorescent intensity of cells incubated with fluorophore labeled virus. B4GalNT2 protein was confirmed to be expressed in the transduced cells, MDCK-B4GalNT2. MDCK-B4GalNT2 bound significantly weaker by MAA1 and MAA2, without a detectable change in SNA binding. Wild type MDCK showed negative staining with DBA and CT-1 antibody, but MDCK-B4GalNT2 were stained strongly positive. This result suggested that transduction of B4GalNT2 converted surface α2,3-sialic acid receptors to Sda-like antigens without affecting the content ofα2,6-sialic acid receptors. MDCK-B4GalNT2 were infected with PR8 and its PB2 mutants. Viral titer was found to be dropped in MDCK-B4GalNT2 with minimal interference in viral polymerase activity. A panel of influenza viruses selected was used to infect MDCK-B4GalNT2. Viral titers and proportion of infected cells by some strains of virus were depleted in MDCK-B4GalNT2 cells in single cycle growth kinetic assay. The differences in viral titers were less significant in multiple cycle growth. MDCK-B4GalNT2 were also infected less efficiently by H5 HA pseudo-particles. Despite the inhibition effect in MDCK-B4GalNT2 to some strains of virus, there were no correlations with the viral receptor binding preferences. For strains that were inhibited, viral attachments were depleted in MDCK-B4GalNT2. The contrary was found to be true for viruses not inhibited in MDCK-B4GalNT2. As a conclusion, overexpression of B4GalNT2 gene in MDCK cells inhibited infection of some strains of influenza virus during viral attachment. MDCK-B4GalNT2 can act as a novel cell model with reduced surface α2,3-sialic acid receptor expression. Influenza viral infection was shown to be interfered by alteration of glycosyltransferases expressions. The results suggested that receptor binding preferences of the same virus in glycan array and on cell surface may differ. | - |
dc.language | eng | - |
dc.publisher | The University of Hong Kong (Pokfulam, Hong Kong) | - |
dc.relation.ispartof | HKU Theses Online (HKUTO) | - |
dc.rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works. | - |
dc.rights | This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. | - |
dc.subject.lcsh | Cell lines | - |
dc.subject.lcsh | Influenza viruses | - |
dc.title | Effect of overexpression of B4GalNT2 in MDCK on influenza viral infection | - |
dc.type | PG_Thesis | - |
dc.description.thesisname | Master of Philosophy | - |
dc.description.thesislevel | Master | - |
dc.description.thesisdiscipline | Pathology | - |
dc.description.nature | published_or_final_version | - |
dc.identifier.doi | 10.5353/th_991044058295203414 | - |
dc.date.hkucongregation | 2018 | - |
dc.identifier.mmsid | 991044058295203414 | - |