An Improved Method for Quantification of Infectious Ebola virus in Dilute Aerosols



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While human-to-human transmission of Ebola virus is thought to occur primarily through direct contact with infected patients or their bodily fluids, short-range aerosol transmission remains a possibility, particularly in health-care settings. Previous studies have demonstrated that the infectious dose of Ebola virus is low when inhaled by nonhuman primates. However, difficulty quantifying low concentrations of infectious Ebola virus in air samples complicates studies assessing the potential public health hazard associated with aerosols containing the virus. In light of this problem, the aim of the present study was to improve sampling and quantification methods for low concentrations of Ebola virus in aerosols. Five low-flow aerosol samplers were assessed for physical and biological sampling efficiencies with aerosols of Ebola virus, with the results demonstrating that gelatin filters were the most efficient sampling method overall and provided improvements in ease-of-use and safety in a biocontainment environment when compared with liquid impingers. Additionally, a microtitration assay using a fluorescent reporter cell line was found to have equivalent sensitivity to several other methods for quantifying viral titers. This method could be automated, which improved throughput and minimized subjectivity. Unlike other assays, this assay is also specific, enabling viral infection to be distinguished from other sources of cellular pathology, and facilitated recovery of gelatin filters in smaller liquid volumes than would otherwise be possible due to assay interference from high gelatin concentrations. This resulted in a more concentrated sample and an increased likelihood of detecting infectious virus in dilute samples. The combined sampling and assay improvements identified in this study resulted in an improvement in sensitivity of approximately ten-fold relative to other published methods for quantifying Ebola virus aerosols, and lowered limits of both quantification and detection. These improved methods can be utilized to improve future studies examining the disease presentation and medical countermeasure efficacy in inhalational animal models of Ebola virus disease, detection or high-confidence non-detection of infectious virus in the air surrounding infected patients and/or animal models, and assessment of the environmental fate of the virus in aerosols.