Rapid Determination of Ebolavirus Infectivity in Clinical Samples Using a Novel Reporter Cell Line

Abstract

Modern ebolavirus diagnostics rely primarily on quantitative reverse transcription-polymerase chain reaction (qRT-PCR), a sensitive method to detect viral genetic material in the acute phase of the disease. However, qRT-PCR does not confirm presence of infectious virus, presenting limitations in patient and outbreak management. Attempts to isolate infectious virus rely on in vivo or basic cell culture approaches, which prohibit rapid results and screening. In this study, we present a novel reporter cell line capable of detecting live ebolaviruses. These cells permit sensitive, large-scale screening and titration of infectious virus in experimental and clinical samples, independent of ebolavirus species and variant.

Keywords: Ebola; infectivity; reporter cell line; virus isolation.

Figure 1

Ebola virus minigenome schematic and reporter cell activation by different filoviruses. (A) Schematic presentation of Ebola virus genome and the minigenome cassette. (B) Activation of zsGreen (ZSG) reporter upon infection. Vero-E6 cells and Vero-Ebola-reporter cells were infected at multiplicity of infection = 0.5 and immunostained 3 days postinfection. Abbreviations: CMV prom., cytomegalovirus promoter; GPC, glycoprotein precursor; HDVRz, hepatitis delta virus ribozyme; HHRz, hammerhead ribozyme; L, ribonucleic acid (RNA)-dependent-RNA-polymerase; NP, nucleoprotein; UTR, untranslated region; VP, viral protein; ZSG, zsGreen; α-Virus, immunostaining against the virus with specific antibodies.

Figure 2

Quantifying infectious virus with the reporter cell line. All inserts represent virus titer values as 50% tissue culture infectious dose (TCID)₅₀/mL. (A) Stock virus titration. (B) Ethylenediaminetetraacetic acid blood and serum samples spiked with Ebola or Sudan viruses at 2 concentrations. (C) Clinical samples. Green bars represent titers determined by zsGreen (ZSG) signal. Orange and red bars represent titers determined by immunostaining on Vero-Ebola-reporter cells and Vero-E6 cells, respectively. Day postinfection is depicted by numbers, and positive (all repeats) or negative results are depicted by +/− symbols. Dashed horizontal lines indicate TCID₅₀ quantification limit. Open bars below quantification limit represent time points at which live virus was detected in all repeat experiments, but at least 1 repeat did not produce enough signal for TCID₅₀ calculation. A and B represent averages and standard deviations from 3 independent experiments. C presents a single experiment. Abbreviation: DRC, Democratic Republic of the Congo.