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Second generation of pseudotype-based serum neutralization assay for Nipah virus antibodies: Sensitive and high-throughput analysis utilizing secreted alkaline phosphatase

2011; Elsevier BV; Volume: 179; Issue: 1 Linguagem: Inglês

10.1016/j.jviromet.2011.11.003

1879-0984

Yoshihiro Kaku, Akira Noguchi, Glenn A. Marsh, Jennifer Barr, Akiko Okutani, Kozue Hotta, Bazartseren Boldbaatar, Shuetsu Fukushi, Christopher C. Broder, Akio Yamada, Satoshi Inoue, Lin‐Fa Wang,

Animal Virus Infections Studies

Nipah virus (NiV), Paramyxoviridae, Henipavirus, is classified as a biosafety level (BSL) 4 pathogen, along with the closely related Hendra virus (HeV). A novel serum neutralization test was developed for measuring NiV neutralizing antibodies under BSL2 conditions using a recombinant vesicular stomatitis virus (VSV) expressing secreted alkaline phosphatase (SEAP) and pseudotyped with NiV F/G proteins (VSV–NiV-SEAP). A unique characteristic of this novel assay is the ability to obtain neutralization titers by measuring SEAP activity in supernatant using a common ELISA plate reader. This confers a remarkable advantage over the first generation of NiV-pseudotypes expressing green fluorescent protein or luciferase, which require expensive and specific measuring equipment. Using panels of NiV- and HeV-specific sera from various species, the VSV–NiV-SEAP assay demonstrated neutralizing antibody status (positive/negative) consistent with that obtained by conventional live NiV test, and gave higher antibody titers than the latter. Additionally, when screening sixty-six fruit bat sera at one dilution, the VSV–NiV-SEAP assay produced identical results to the live NiV test and only required a very small amount (2 μl) of sera. The results suggest that this novel VSV–NiV-SEAP assay is safe, useful for high-throughput screening of sera using an ELISA plate reader, and has high sensitivity and specificity.