Quantitative mutant analysis of viral quasispecies by chip-based matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry

Abstract

RNA viruses exist as quasispecies, heterogeneous and dynamic mixtures of mutants having one or more consensus sequences. An adequate description of the genomic structure of such viral populations must include the consensus sequence(s) plus a quantitative assessment of sequence heterogeneities. For example, in quality control of live attenuated viral vaccines, the presence of even small quantities of mutants or revertants may indicate incomplete or unstable attenuation that may influence vaccine safety. Previously, we demonstrated the monitoring of oral poliovirus vaccine with the use of mutant analysis by PCR and restriction enzyme cleavage (MAPREC). In this report, we investigate genetic variation in live attenuated mumps virus vaccine by using both MAPREC and a platform (DNA MassArray) based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Mumps vaccines prepared from the Jeryl Lynn strain typically contain at least two distinct viral substrains, JL1 and JL2, which have been characterized by full length sequencing. We report the development of assays for characterizing sequence variants in these substrains and demonstrate their use in quantitative analysis of substrains and sequence variations in mixed virus cultures and mumps vaccines. The results obtained from both the MAPREC and MALDI-TOF methods showed excellent correlation. This suggests the potential utility of MALDI-TOF for routine quality control of live viral vaccines and for assessment of genetic stability and quantitative monitoring of genetic changes in other RNA viruses of clinical interest

Microarray analysis of evolution of RNA viruses: Evidence of circulation of virulent highly divergent vaccine-derived polioviruses

“…Our previous studies demonstrating a correlation between minor sequence heterogeneity and neurovirulence of OPV (13,(42)(43)(44) and plasticity of the genome of attenuated mumps virus (45,46) have led us to propose a molecular consistency approach to monitoring production of live viral vaccines (47). Mutant analysis by PCR and the restriction enzyme cleavage method we proposed for this purpose were accepted by the World Health Organization as a standard quality control test of OPV (48).…”

MALDI-TOF MS in Prenatal Genomics

“…hME-based MALDI-TOF MS can be used to analyse copy number variation of each allele [45,82,83]. An allele-specific primer extension reaction designed by the hME to a heterozygous SNP generates two extension products, each has unique molecular weight, resulting in 2 peaks on MALDI- The relative quantification of copy number can be estimated through dividing the peak intensity generated from one allele by that of another allele.…”

Genotyping single nucleotide polymorphisms by mass spectrometry

“…The analyte is robotically transferred to silicon chips that are precharged with matrix (SpectroCHIPS TM ). The MassEXTEND TM assay has been applied to a large-scale study of over 9,000 gene-based SNPs in pooled DNA samples (Buetow et al, 2001), to the identification of differences between bacterial strains (Amexis et al, 2001) and numerous others studies (Cashman et al, 2001;Nakai et al, 2002).…”