Allele-specific qRT-PCR demonstrates superior detection of single nucleotide polymorphisms as genetic markers for West Nile virus compared to Luminex® and quantitative sequencing
Introduction
West Nile virus (WNV) is a single-stranded, positive-sense RNA virus belonging to the family Flaviviridae. The WNV genome is approximately 11,000 nucleotides in length and encodes a single polyprotein that is cleaved into three structural proteins (C, prM, E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) (Rice et al., 1985).
As an arthropod-borne virus, WNV naturally perpetuates in a two-host transmission cycle between birds and Culex mosquitoes with occasional tangential infection of humans and other vertebrates. To maintain sufficient fitness in both insect and vertebrate hosts, the genome of flaviviruses seems to be conserved, likely due to alternation between host and vector, and purifying selection in the avian host (Jerzak et al., 2008, Coffey et al., 2008). When mutations occur, they may be detrimental and lost rapidly, neutral and maintained, or beneficial by altering significantly the phenotype and being selected positively (Domingo et al., 1996).
Direct sequencing is the method of choice to identify nucleotide substitutions, study phylogenetic relationships and determine genotypic evolution. However, relating genotypic variation to significant phenotypic change is difficult and often requires intensive reverse-engineering experiments. Alternatively, to investigate phenotypic change, in vitro and in vivo fitness competition experiments may be used to compare the replicative capacity of two viruses concurrently competing in the same host (Weaver et al., 1999). Such studies can be used to compare field isolates to a reference or founding strain while controlling for inter-host variability. However, the high degree of genetic conservation among WNV isolates complicates the development of genotype-specific primers and probes for RT-PCR. Therefore, a phenotypically neutral genetic marker is required to label the reference strain for competition against wildtype isolates of interest. The analysis of samples mixed with two genetically similar viral populations that may have markedly different titers is challenging as it requires highly specific and quantitative detection based on one or a few nucleotide differences.
The aim of the current study was to engineer a WNV reference strain containing a stable and fitness neutral genetic marker that would facilitate developing quantitative and specific detection methods to track concurrently genetically marked and wildtype viruses in competition assays. Three strains of WNV were marked genetically by site-directed mutagenesis of either one or five synonymous nucleotide substitutions in the E gene between nucleotide positions 2449–2454 to serve as reference viruses for in vitro and in vivo fitness competition studies. In an effort to develop a suitable detection method, three approaches were compared: Luminex® technology, quantitative sequencing and quantitative real-time RT-PCR.
The Luminex® xTAG® protocol uses a liquid suspension microarray platform to detect individually sequence-tagged, color-coded microspheres with a flow cytometric laser detection system (Luminex® Corporation, Austin, TX, USA). It allows for high-throughput, multiplex testing for as many as 100 different nucleic acid sequences in one sample and previously has been used for host identification of Culex mosquito blood meals (Thiemann et al., 2012) and for detection of several subtypes of influenza A viruses (Smith et al., 2012). While one laser interrogates microsphere identity, the other quantifies the fluorescence derived from the biotinylated template resulting in a cumulative signal used for template quantitation.
Quantitative sequencing is used to measure quantitatively multiple single nucleotide polymorphisms from sequencing chromatograms (Hall and Little, 2007). The peak area and/or height from individual, overlapping (polymorphic) sequence chromatograms is measured with PHRED software and PolySNP, a PERL script, and utilized to estimate the relative template frequency in pooled DNA samples based on the proportions of the overlapping peaks (Hall and Little, 2007). This approach previously has been used in fitness competitions of WNV (Fitzpatrick et al., 2010, Deardorff et al., 2011) and dengue virus (Groat-Carmona et al., 2012).
Finally, TaqMan® (Applied Biosystems [ABI], Foster City, California, USA) is a quantitative one-step real-time RT-PCR approach that can be multiplexed and is used to distinguish closely related RNA templates with sequence-specific primer and probe sets. A specific single nucleotide polymorphism can be targeted using individual allele-specific TaqMan® probes containing distinct fluorescent dyes and primer sets that uniquely align with the genome providing high specificity for the allele of interest (ABI, USA).
In the current study, Luminex® technology, quantitative sequencing and TaqMan® qRT-PCR were compared with regard to their specificity and ability to accurately quantify a wide range of titers of wildtype and mutant WNV RNA in mixed samples during co-infection experiments. Each method is described and the most successful approach, a novel singleplex quantitative qRT-PCR assay using a sequence-specific reverse primer set is presented.
Section snippets
Viruses and construction of mutants
Plasmids containing WNV genomic information from three wildtype strains of WNV were used to engineer mutants containing either one or five synonymous nucleotide (nt) substitutions in the carboxy terminus area of the E gene (nt 967–2469). A previously generated infectious clone-derived virus based on a field isolate of the NY99 strain (Kinney et al., 2006) was marked genetically by either a single nucleotide transition from C to T at position nt 2454 (1nt-mutation) or by five-nucleotide
Luminex xTAG® microsphere array
Equal concentrations of NY99, NY99-1nt and NY99-5nt were transcribed and PCR amplified singly with either WNV.2296.F/WNV.2786.R or WNV.2387.F/WNV.2530.R (Table 2). Transcripts amplified with primer set WNV.2296.F/WNV.2786.R resulted in MFI values between 5 and 60 which were below the MFI expected for each microsphere (Table 4). In contrast, the use of WNV.2387.F/WNV.2530.R resulted in higher MFI values of 2342 and 4311 for LUA10/75 and a correct MFI quantification of LUA50/40 with 5528 and
Discussion
Our study introduces a novel approach to detect single nucleotide polymorphisms (SNPs) as genetic markers in the envelope gene region of WNV using different reverse primers for allele-specific RT-PCR quantitation. Other approaches including Luminex xTAG® microsphere array and quantitative sequencing were evaluated for their ability to quantitatively distinguish the engineered mutant viruses from wildtype WNV.
The substitution of five-nucleotides (CTCTCC → TTGAGT) at positions 2449 and 2451–2454
Conflict of interest statement
None.
Acknowledgments
The authors acknowledge Dr. Brent Ewing for providing an academic license of PolySNP and PHRED. Special thanks to Dr. Gregory Ebel and Dr. Gerod Hall for their advice on the implementation of the quantitative sequencing. G. Worwa was supported by the Swiss National Science Foundation (SNSF; PBBEP3_128345) and the Swiss Foundation for Grants in Biology and Medicine (SFGBM; PASMP3_137034/1). C. Andrade was supported by the National Institutes of Health training grant (NIH; T32 AI074550). Funding
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