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

RNA viruses exist as quasispecies, heterogeneous and dynamic mixtures of mu tants having one or more consensus sequences. An adequate description of th e genomic structure of such viral populations must include the consensus se quence(s) plus a quantitative assessment of sequence heterogeneities. For e xample, in quality control of live attenuated viral vaccines, the presence of even small quantities of mutants or revertants may indicate incomplete o r unstable attenuation that may influence vaccine safety. Previously, we de monstrated 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 l aser desportion/ionization time-of-flight (MALDI-TOF) mass spectrometry. Mu mps 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 characte rizing sequence variants in these substrains and demonstrate their use in q uantitative analysis of substrains and sequence variations in mixed virus c ultures and mumps vaccines. The results obtained from both the MAPREC and M ALDI-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.