A rapid and quantitative assay for measuring antibody-mediated neutralization of West Nile virus infection

Abstract

West Nile virus (WNV) is a neurotropic flavivirus within the Japanese encephalitis antigenic complex that is responsible for causing West Nile encephalitis in humans. The surface of WNV virions is covered by a highly ordered icosahedral array of envelope proteins that is responsible for mediating attachment and fusion with target cells. These envelope proteins are also primary targets for the generation of neutralizing antibodies in vivo. In this study, we describe a novel approach for measuring antibody-mediated neutralization of WNV infection using virus-like particles that measure infection as a function of reporter gene expression. These reporter virus particles (RVPs) are produced by complementation of a sub-genomic replicon with WNV structural proteins provided in trans using conventional DNA expression vectors. The precision and accuracy of this approach stem from an ability to measure the outcome of the interaction between antibody and viral antigens under conditions that satisfy the assumptions of the law of mass action as applied to virus neutralization. In addition to its quantitative strengths, this approach allows the production of WNV RVPs bearing the prM-E proteins of different WNV strains and mutants, offering considerable flexibility for the study of the humoral immune response to WNV in vitro. WNV RVPs are capable of only a single round of infection, can be used under BSL-2 conditions, and offer a rapid and quantitative approach for detecting virus entry and its inhibition by neutralizing antibody.

Genetic and Phenotypic Properties of Vero Cell-Adapted Japanese Encephalitis Virus SA14-14-2 Vaccine Strain Variants and a Recombinant Clone, Which Demonstrates Attenuation and Immunogenicity in Mice

“…46,53 had a 10-fold higher IC LD 50 and mice had a significantly longer, 8 days, median survival time (Table 5 and Figure 3). Similarly, JEV SA14-14-2 variant D differed from variant H by six reversion mutations (Table 1), and had a 17-fold higher IC LD 50 and mice had an 8-day median survival time (Table 5 and Figure 3). The most attenuated virus was JEV SA14-14-2 variant C, which differed by two unique mutations from variant D ( Table 1) that increased the IC LD 50 level 60-fold and 80% of mice survived a 10 2 PFU dose (Table 5 and Figure 3).…”

“…47 The JEV SA14-14-2 RVPs expressing green fluorescent protein (GFP) were produced in HEK 293T cells, as described previously. 47,50,51 The RVP titers were determined by serial dilution of JEV RVP stocks and infecting Raji cells in 96-well plates. The RVP-infected cells were fixed with paraformaldehyde and the percentage of GFP expressing cells was determined by flow cytometry.…”

“…47 The data were analyzed using GraphPad Prism (GraphPad Software, La Jolla, CA) and were fit by a doseresponse curve (variable slope) to determine the 50% effective neutralization concentration (EC 50 ). An immunized mouse seroconverted if its serum had a detectable EC 50 . Titers are reported as the reciprocal geometric mean EC 50 titer.…”

“…An immunized mouse seroconverted if its serum had a detectable EC 50 . Titers are reported as the reciprocal geometric mean EC 50 titer.…”

Axonal transport mediates West Nile virus entry into the central nervous system and induces acute flaccid paralysis

“…To test whether WNV neuron-to-target-cell spread occurred in the absence of viral packing and egress, primary neurons were infected with pseudotyped WNV reporter virus particles (RVP). RVP deliver WNV replicons that encode GFP but lack structural proteins and thus are only capable of a single round of cellular infection (16). Notably, no increase in the numbers of GFP-positive cells was observed in RVP-infected cortical neuron cultures over time (SI Fig.…”