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The Susceptibilities of Respiratory Syncytial Virus to Nucleolin Receptor Blocking and Antibody Neutralization are Dependent upon the Method of Virus Purification

Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB T6G 2R3, Canada
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Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Academic Editor: Curt Hagedorn
Viruses 2017, 9(8), 207; https://doi.org/10.3390/v9080207
Received: 23 June 2017 / Revised: 25 July 2017 / Accepted: 27 July 2017 / Published: 3 August 2017
(This article belongs to the Section Antivirals & Vaccines)
Respiratory Syncytial Virus (RSV) that is propagated in cell culture is purified from cellular contaminants that can confound experimental results. A number of different purification methods have been described, including methods that utilize fast protein liquid chromatography (FPLC) and gradient ultracentrifugation. Thus, the constituents and experimental responses of RSV stocks purified by ultracentrifugation in sucrose and by FPLC were analyzed and compared by infectivity assay, Coomassie stain, Western blot, mass spectrometry, immuno-transmission electron microscopy (TEM), and ImageStream flow cytometry. The FPLC-purified RSV had more albumin contamination, but there was less evidence of host-derived exosomes when compared to ultracentrifugation-purified RSV as detected by Western blot and mass spectrometry for the exosome markers superoxide dismutase [Cu-Zn] (SOD1) and the tetraspanin CD63. Although the purified virus stocks were equally susceptible to nucleolin-receptor blocking by the DNA aptamer AS1411, the FPLC-purified RSV was significantly less susceptible to anti-RSV polyclonal antibody neutralization; there was 69% inhibition (p = 0.02) of the sucrose ultracentrifugation-purified RSV, 38% inhibition (p = 0.03) of the unpurified RSV, but statistically ineffective neutralization in the FPLC-purified RSV (22% inhibition; p = 0.30). The amount of RSV neutralization of the purified RSV stocks was correlated with anti-RSV antibody occupancy on RSV particles observed by immuno-TEM. RSV purified by different methods alters the stock composition and morphological characteristics of virions that can lead to different experimental responses. View Full-Text
Keywords: respiratory syncytial virus; fast protein liquid chromatography; purification; sucrose gradient ultracentrifugation; density; mass spectrometry; transmission electron microscopy; antibody neutralization; nucleolin respiratory syncytial virus; fast protein liquid chromatography; purification; sucrose gradient ultracentrifugation; density; mass spectrometry; transmission electron microscopy; antibody neutralization; nucleolin
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Bilawchuk, L.M.; Griffiths, C.D.; Jensen, L.D.; Elawar, F.; Marchant, D.J. The Susceptibilities of Respiratory Syncytial Virus to Nucleolin Receptor Blocking and Antibody Neutralization are Dependent upon the Method of Virus Purification. Viruses 2017, 9, 207.

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