Abstract
BackgroundThe evolution of viral quasispecies can influence viral pathogenesis and the response to antiviral treatments. Mutant clouds in infected organisms represent the first stage in the genetic and antigenic diversification of RNA viruses, such as foot and mouth disease virus (FMDV), an important animal pathogen. Antigenic variants of FMDV have been classically diagnosed by immunological or RT-PCR-based methods. DNA microarrays are becoming increasingly useful for the analysis of gene expression and single nucleotide polymorphisms (SNPs). Recently, a FMDV microarray was described to detect simultaneously the seven FMDV serotypes. These results encourage the development of new oligonucleotide microarrays to probe the fine genetic and antigenic composition of FMDV for diagnosis, vaccine design, and to gain insight into the molecular epidemiology of this pathogen.ResultsA FMDV microarray was designed and optimized to detect SNPs at a major antigenic site of the virus. A screening of point mutants of the genomic region encoding antigenic site A of FMDV C-S8c1 was achieved. The hybridization pattern of a mutant includes specific positive and negative signals as well as crosshybridization signals, which are of different intensity depending on the thermodynamic stability of each probe-target pair. Moreover, an array bioinformatic classification method was developed to evaluate the hybridization signals. This statistical analysis shows that the procedure allows a very accurate classification per variant genome.ConclusionA specific approach based on a microarray platform aimed at distinguishing point mutants within an important determinant of antigenicity and host cell tropism, namely the G-H loop of capsid protein VP1, was developed. The procedure is of general applicability as a test for specificity and discriminatory power of microarray-based diagnostic procedures using multiple oligonucleotide probes.
Highlights
The evolution of viral quasispecies can influence viral pathogenesis and the response to antiviral treatments
We report assay conditions that have been optimized to detect the presence of several point mutants at this major antigenic site of foot and mouth disease virus (FMDV), and develop a support vector machine (SVM)based procedure to automatize sample classification hybridization intensities and to set up limits for reliable diagnosis
Specificity and sensitivity optimization of FMDV microarray In a first approach, 8 DNA oligonucleotides were designed for the set up of an FMDV microarray
Summary
The evolution of viral quasispecies can influence viral pathogenesis and the response to antiviral treatments. Mutant clouds in infected organisms represent the first stage in the genetic and antigenic diversification of RNA viruses, such as foot and mouth disease virus (FMDV), an important animal pathogen. A FMDV microarray was described to detect simultaneously the seven FMDV serotypes These results encourage the development of new oligonucleotide microarrays to probe the fine genetic and antigenic composition of FMDV for diagnosis, vaccine design, and to gain insight into the molecular epidemiology of this pathogen. The control of diseases associated with highly variable RNA viruses requires close monitoring of the variant virus types that periodically dominate in viral populations. This is due to high mutation rates, quasispecies dynamics and population bottlenecks that often accompany virus transmission [reviewed in [1]]. Methodology to discern among minor variants of the same viral genotype or serotype is essential for epidemiological surveillance and the planning of disease control strategies
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