UBC Theses and Dissertations
Application of MagLev-based isolation technology for the rapid and sensitive concentration of influenza viruses Khalid, Muhammad Faizan
Viruses are a fundamental branch of the tree of life. They are responsible for several biological processes and a major source of human and plant diseases. Despite their relevance to life however, most techniques to study them are dependent on indirect and invasive methods like PCR or immunological and cytological assays which require large numbers of whole viruses to function reliably. This is also true for the influenza virus which has caused several pandemics in human history. Many established techniques in virology additionally require extensive storage and intermediary steps to preserve and increase viral titters for analysis. These steps impose constraints on the process of detection and isolation while also destroying virus particles. To reduce the constraints imposed by things like primer bias, storage conditions and virus host interactions during enumeration steps, in this thesis I present a Magnetic Levitation (MagLev) based technique for the isolation and detection of influenza viruses. My main objective was to assess the performance of Magnetic Levitation (MagLev) based techniques for the sensitive and rapid isolation of intact influenza viruses in biological samples. The results show that influenza virions are levitated within a MagLev systems column consistently and distinctly using either Gadobutrol or Super Paramagnetic Iron Oxide Nanoparticle (SPION) solutions (in the MagLev) but do not produce a reliable visible marker for identification. However, the pure influenza A viral stock used in the experiment did produce a visualization in two trials which was used to determine a first ever direct MagLev based estimate of influenza A viral density (~ 0.978 ± 0.02 g/cm³). Which is notably lower than previous estimates of viral density (1.014-1.265 g/cm³) obtained through sucrose gradient based ultracentrifugation. Additionally, the results of viral quantification via Q-RTPCR and Tissue Culture Infectious Dose 50 (TCID50) assays corroborated the evidence of viral levitation. Both validation methods produced relatively consistent results, wherein fractions of the MagLev systems column produced a lower Ct value or higher TCID50/ml indicating a higher presence of intact influenza A virus in regions of the MagLev column as a consequence of levitation. This work shows that influenza virions can be levitated reliably and non-invasively using a MagLev based apparatus, thus opening up room for future work using the technology. With MagLev based techniques, we can develop novel ways of collecting, studying and concentrating viral samples that reduce the constraints imposed by traditional virology techniques.
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