Allele-specific qRT-PCR demonstrates superior detection of single nucleotide polymorphisms as genetic markers for West Nile virus compared to Luminex® and quantitative sequencing

Abstract

To enable in vivo and in vitro competitive fitness comparisons among West Nile viruses (WNV), three reference viruses were genetically marked by site-directed mutagenesis with five synonymous nucleotide substitutions in the envelope gene region of the genome. Phenotypic neutrality of the mutants was experimentally assessed by competitive replication in cell culture and genetic stability of the substituted nucleotides was confirmed by direct sequencing. Luminex® technology, quantitative sequencing and quantitative RT-PCR (qRT-PCR) were compared in regard to specificity, sensitivity and accuracy for quantitation of wildtype and genetically marked viruses in mixed samples based on RNA obtained from samples of known viral titers. While Luminex® technology and quantitative sequencing provided semi-quantitative or qualitative measurements a sequence-specific primer extension approach using a specific reverse primer set in singleplex qRT-PCR demonstrated the best quantitation and specificity in the detection of RNA from wildtype and mutants viruses.

Comparing Competitive Fitness of West Nile Virus Strains in Avian and Mosquito Hosts

“…Using an infectious clone-derived virus (COAV997-IC) of the founding California COAV997-2003 isolate [ 11 ], we previously generated the genetically marked COAV997-5nt mutant utilized in the current in vivo fitness competition study [ 24 ]. The COAV997-5nt contains five nucleotide substitutions ( C T CTCC → T T GAGT ) in the envelope gene at nucleotide positions 2449 and 2451–2454 [ 24 ].…”

“…Using an infectious clone-derived virus (COAV997-IC) of the founding California COAV997-2003 isolate [ 11 ], we previously generated the genetically marked COAV997-5nt mutant utilized in the current in vivo fitness competition study [ 24 ]. The COAV997-5nt contains five nucleotide substitutions ( C T CTCC → T T GAGT ) in the envelope gene at nucleotide positions 2449 and 2451–2454 [ 24 ]. Both COAV997-IC and COAV997-5nt demonstrated indistinguishable growth profiles in Vero and C6/36 cells over the course of three passages and yielded similar RNA copy numbers indicating fitness neutrality in vitro [ 24 ].…”

“…The COAV997-5nt contains five nucleotide substitutions ( C T CTCC → T T GAGT ) in the envelope gene at nucleotide positions 2449 and 2451–2454 [ 24 ]. Both COAV997-IC and COAV997-5nt demonstrated indistinguishable growth profiles in Vero and C6/36 cells over the course of three passages and yielded similar RNA copy numbers indicating fitness neutrality in vitro [ 24 ]. A quantitative RT-PCR (qRT-PCR) assay using specific reverse primers was developed to distinguish between RNA from wildtype COAV997-IC and COAV997-5nt in mixed competition experiments [ 24 ].…”

“…Both COAV997-IC and COAV997-5nt demonstrated indistinguishable growth profiles in Vero and C6/36 cells over the course of three passages and yielded similar RNA copy numbers indicating fitness neutrality in vitro [ 24 ]. A quantitative RT-PCR (qRT-PCR) assay using specific reverse primers was developed to distinguish between RNA from wildtype COAV997-IC and COAV997-5nt in mixed competition experiments [ 24 ]. This assay showed a linear dynamic detection range of at least 6 log 10 RNA copies with almost identical amplification efficiencies between wildtype and COAV997-5nt and a calculated detection limit of 250 RNA copies [ 24 ].…”

“…A quantitative RT-PCR (qRT-PCR) assay using specific reverse primers was developed to distinguish between RNA from wildtype COAV997-IC and COAV997-5nt in mixed competition experiments [ 24 ]. This assay showed a linear dynamic detection range of at least 6 log 10 RNA copies with almost identical amplification efficiencies between wildtype and COAV997-5nt and a calculated detection limit of 250 RNA copies [ 24 ].…”