Variability in dengue titer estimates from plaque reduction neutralization tests poses a challenge to epidemiological studies and vaccine development.

BACKGROUND: Accurate determination of neutralization antibody titers supports epidemiological studies of dengue virus transmission and vaccine trials. Neutralization titers measured using the plaque reduction neutralization test (PRNT) are believed to provide a key measure of immunity to dengue viruses, however, the assay's variability is poorly understood, making it difficult to interpret the significance of any assay reading. In addition there is limited standardization of the neutralization evaluation point or statistical model used to estimate titers across laboratories, with little understanding of the optimum approach. METHODOLOGY/PRINCIPAL FINDINGS: We used repeated assays on the same two pools of serum using five different viruses (2,319 assays) to characterize the variability in the technique under identical experimental conditions. We also assessed the performance of multiple statistical models to interpolate continuous values of neutralization titer from discrete measurements from serial dilutions. We found that the variance in plaque reductions for individual dilutions was 0.016, equivalent to a 95% confidence interval of 0.45-0.95 for an observed plaque reduction of 0.7. We identified PRNT75 as the optimum evaluation point with a variance of 0.025 (log10 scale), indicating a titer reading of 1∶500 had 95% confidence intervals of 1∶240-1∶1000 (2.70±0.31 on a log10 scale). The choice of statistical model was not important for the calculation of relative titers, however, cloglog regression out-performed alternatives where absolute titers are of interest. Finally, we estimated that only 0.7% of assays would falsely detect a four-fold difference in titers between acute and convalescent sera where no true difference exists. CONCLUSIONS: Estimating and reporting assay uncertainty will aid the interpretation of individual titers. Laboratories should perform a small number of repeat assays to generate their own variability estimates. These could be used to calculate confidence intervals for all reported titers and allow benchmarking of assay performance

A human PrM antibody that recognizes a novel cryptic epitope on dengue E glycoprotein

10.1371/journal.pone.0033451PLoS ONE74

Serological evidence for transmission of multiple dengue virus serotypes in Papua New Guinea and West Papua prior to 1963

Little is known about the natural history of dengue in Papua New Guinea (PNG). We assessed dengue virus (DENV)-specific neutralizing antibody profiles in serum samples collected from northern and southern coastal areas and the highland region of New Guinea between 1959 and 1963. Neutralizing antibodies were demonstrated in sera from the northern coast of New Guinea: from Sabron in Dutch New Guinea (now known as West Papua) and from four villages in East Sepik in what is now PNG. Previous monotypic infection with DENV-1, DENV-2, and DENV-4 was identified, with a predominance of anti-DENV-2 neutralizing antibody. The majority of positive sera demonstrated evidence of multiple previous DENV infections and neutralizing activity against all four serotypes was detected, with anti-DENV-2 responses being most frequent and of greatest magnitude. No evidence of previous DENV infection was identified in the Asmat villages of the southern coast and a single anti-DENV-positive sample was identified in the Eastern Highlands of PNG. These findings indicate that multiple DENV serotypes circulated along the northern coast of New Guinea at different times in the decades prior to 1963 and support the notion that dengue has been a significant yet neglected tropical infection in PNG for many decades

Comparative analysis of the NS 1 gene sequences of dengue-1 viruses prototype Hawaii strain and Thai isolate TH-Sman, and determination of the intratypic variation of NS 1 protein among dengue-1 viruses

In view of a previous report on significant antigenic and biophysical differences between the purified soluble complement-fixing antigens of dengue-1 virus strains Hawaii and TH-Sman, the NS 1 genes of both virus isolates were cloned, sequenced, and compared in an attempt to define the genetic basis for the observed differences. Sequence comparison revealed ten encoded amino acid differences between the NS 1 genes of both viruses. Three of these amino acid differences, which are associated with a change in charge distribution, are clustered within the major antigenic region previously defined by studies of recombinant dengue-1 NS 1 protein expressed in E. coli. In parallel, the NS 1 sequences of both Hawaii and TH-Sman isolates were also aligned and compared with two other published dengue-1 NS 1 protein sequences to determine the intratypic variation of dengue-1 NS 1 antigen. Pairwise comparisons between the encoded amino acid sequences revealed a variability of 1.1% to 3.1% difference in the NS 1 protein among dengue-1 strains, which is comparable to that reported for dengue-1 envelope protein (0.2% to 3.6% difference) but less than that of dengue-2 NS 1 protein (0.6% to 7.4% difference). © 1993 Springer-Verlag.link_to_subscribed_fulltex