Genome-wide real-time PCR for West Nile virus reduces the false-negative rate and facilitates new strain discovery

Abstract

West Nile virus (WNV) causes significant morbidity and mortality worldwide. Transplant and transfusion recipients as well as the elderly are particularly at risk. WNV shows strain variation from season to season and from locale to locale. This poses a significant problem for diagnosis. Most assays use a single primer pair to detect WNV by QPCR, and can fail to detect novel stains. To overcome this limitation, a genome-wide, multiple primer-based real-time QPCR assay was developed for WNV. The same assay can be used for quantitation, viral variant discovery as well as for amplification of the entire viral genome using a single annealing temperature. It improves upon routine diagnosis as well as facilitates laboratory investigations of the pathology of WNV.

Figure 1

SNP distribution across the WNV genome. Panel A-D SNP positions are indicated by a black bar. A, distribution across the entire genome; B, distribution across the most 5′ 1,529 nucleotides; C, distribution across the E orf; D, distribution across the most 3′ 1,529 nucleotides. E, plot of the between SNP distances on the vertical against the SNP position on the horizontal axis across the entire 11,029 WNV genome (genbank accession # AF196835). F, Frequency histogram of the distributions of SNPs per genome. G, Distribution of base calls counted as SNP. Letters follow IUBMB nomenclature. R represents purine (A or G) and Y represents pyrimidine (T or C).

Figure 2

Annealing temperature dependence of qPCR for individual primers pair combinations. The vertical axis is reversed as lower CTs indicate a higher target abundance or, as all reactions received the same amount of input target a higher qPCR efficiency. The horizontal axis indicated the annealing temperature. The target was cDNA from WNV strain NY99.

Figure 3

Amplicon length dependence of qPCR. Boxplot of CT values at different annealing temperature (vertical axis) for primer pairs of different length (shown on the horizontal axis). Boxframe indicates the 1 and 3 quartiles, the solid bar the median and whiskers the minima and maxima. Outliers are indicated by circles. Note that the vertical axis is not to scale, but categorical.

Figure 4

Genome-wide qPCR. Picture of an ethidium bromide stained agarose gel yielding same size amplicons (A) or combinations to yield largers amplicons (B). The primer names are indicated above the gel. Note that the primer names do not indicate the map position, which can be obtained from table 1. (C) Shown are the map positions for all primers used in this study using the West Nile virus strain NY99-flamingo382-99 coordinates (genbank entry AF196835). Forward primers are above and reverse primers below the genome coordinates. Primer combinations and amplicon sizes, which were used for sequencing are indicated by vertical lines.

Figure 5

Volume dependence of qPCR. (A) Shown on the horizontal axis is the amount of input cDNA sample and on the vertical axis the mean CT of four replicate measurements after real-time qPCR. Lower CT corresponds to better detection on a ₂log scale. (B) Shown is the standard deviation (SD) in CT on the vertical and input cDNA volume on the horizontal axis.