Simplified plaque reduction neutralization assay for dengue viruses by semimicro methods in BHK-21 cells: comparison of the BHK suspension test with standard plaque reduction neutralization

Abstract

A newly modified semimicro plaque reduction neutralization test (PRNT) in BHK cells was compared with a standard PRNT in bottles with LLC-MK2 monolayers and with an LLC-MK2 PRNT adapted to semimicro methods. The BHK semimicro PRNT compared favorably in terms of sensitivity in detecting dengue antibody (96%), specificity at a screening dilution (95%), and ability to detect seroconversion to dengue viruses of three serotypes (93%). Disagreements between the BHK test and the LLC-MK2 tests were attributed to greater sensitivity of the BHK test in detecting dengue type 2 (DEN-2) antibody in acute-phase sera and to apparent low-level DEN-1I/DEN-3 cross-reactions in some sera in all three tests. The BHK PRNT was easier, faster, and more economical than either of the LLC-MK2 tests. Many of the benefits of the BHK PRNT derive from the fact that cells are infected while still in suspension, at the time of cell splitting, hence the term "BHK suspension test." * Corresponding author. cells as a suspension; hence, we refer to it as the "BHK suspension test.'"

High seroprevalence of antibodies against dengue virus in a prospective study of schoolchildren in Managua, Nicaragua

“…An additional 85 (7%) sample pairs were also processed by plaque reduction neutralisation test (PRNT). All PRNT testing was carried out by the US Naval Medical Research Institute Detachment (NAMRID), Lima, Perú (Morens et al 1985) at a 1:30 and 1:60 dilution for DENV3 and DENV4 and 1:60 and 1:120 dilution for DENV1 and DENV2. Although West Nile Virus (WNV) has not yet been detected in Nicaragua, 82 study samples were analysed by IgM MAC-ELISAs with DEN or WNV antigen (CDC, Fort Collins, CO, USA) in parallel, and the results suggest that our population is naïve to WNV infection (data not shown).…”

In vitro and in vivo studies of the Interferon-alpha action on distinct Orthobunyavirus

“…Medium was removed, serial 10-fold dilutions of viral stocks diluted in MEM with 5% FBS were added (0.2 mL/well) in quadruplicates, and the cells were incubated for 2 h at 37 • C. Subsequently, the viral inoculum was removed, and 1.0 mL of a combination (v/v) of 1% low-melting-point agarose plus 2× MEM (10% FBS) was added to each well; the plates were incubated at 37 • C for 3 days for OROV and GMAV, 5 days for CARV and GROV, and 9 days for TCMV. The plaques were visualized after staining with a naphtol blue black solution (15 min) (Morens et al, 1985), preceded of removal of the agarose plug. The plaques were counted under an inverted microscope, and the virus titer was determined as log 10 PFU mL −1 .…”

A continuous peptide epitope reacting with pandemic influenza AH1N1 predicted by bioinformatic approaches

“…*Statistical analysis of the ELISA, p < 0.05, Pandemic-p1 versus Pandemic-p2, dilution 1:50, one-way ANOVA, Tuckey-Kramer honestly significant difference. antibodies in minor quantity in the infected or vaccinated group (titer 1: 103 and 1: 160, respectively) (Figure 6)[73,74].…”