Pseudoparticle neutralization assay for detecting ebola- neutralizing antibodies in biosafety level 2 settings.

Ebola virus is currently causing an unprecedented outbreak in West Af-rica. The pathogen is classified as a biosafety level 4 (BSL-4) 1 pathogen and should be handled only in high-level biosafety settings (1). Virus neutralization is considered to be the gold standard serological assay for infections, but in the case of Ebola, this test requires culturing of live virus in a BSL-4 facility. Because of these stringent biosafety requirements , options for clinical diagnosis or epidemiological studies of Ebola cases are limited. For this reason, we developed a pseudoviral particle neutralization (PPNT) assay that can detect neutralizing antibody to Ebola virus in BSL-2 containments. Ebola glycoprotein (GP) is a major target of neutralizing antibod-ies in humans. The pseudoviral particle generated in this study is a len-tiviral vector carrying the GP of Zaire ebolavirus detected in the current outbreak (2). Methods for the pseudoviral particle production and PPNT assay were essentially identical to those for Middle East respiratory syndrome (MERS) and influenza , as previously described (3, 4). Briefly, a codon-optimized GP sequence was chemically synthesized (Genscript) and subcloned into a protein-expressing vector, pcDNA3.1ϩ. The resulting pseudo-viral particle is capable of achieving only a single-round infection and contains a luciferase reporter gene that can be expressed in infected cells. Anti-GP neutralizing antibodies can inhibit the entry of this pseudoviral particle into cells, thereby inhibiting the expression of luciferase in the assay. For each Ebola pseudoviral particle production , the preparation was titrated and diluted to a level that could produce 10000 –50000 counts/s in a positive infection control reaction in the lu-ciferase assay (Steady-Glo Luciferase Assay System, Promega). In contrast, mock-infected cells (i.e., cells treated in the absence of pseudoviral particle) yielded background signals in the assay (Ͻ1% of the positive infection control) (Table 1). For a typical PPNT assay for se-rological screening, 5 ␮L heat-treated serum sample (56 °C, 30 min) was in-cubated with the diluted pseudopar-ticles in a 100-␮L reaction at 4 °C for 60 min before inoculation onto Vero cells (3, 4). The luciferase activities of infected cells were examined 72 h postinfection. Based on our previous experiences with similar assays (3, 4), the cutoff value for a positive reaction was set to be 10% of the value deduced from the positive infection control (see above). For determining the neutralization antibody titer of a serum sample, 2-fold serially diluted samples of a specimen (starting dilution 1:20) were used …