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 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.

Correlation of genetic variability with safety of mumps vaccine Urabe AM9 strain.

The Urabe AM9 strain of mumps vaccine live is known for its genetic instability and some vaccines derived from this strain were withdrawn from the market due to an excessive number of vaccine-associated parotitis and meningitis cases. To identify the molecular basis of this instability, we determined complete nucleotide sequences of several stocks of the Urabe strain used for vaccine production by different manufacturers and of two clinical isolates from cases of vaccine-associated meningitis. In contrast to previously published studies relating the Lys335 --> Glu mutation in the viral HN gene with neurovirulence of mumps virus, we could not confirm any association of this mutation with the safety of mumps vaccine. Each of the three vaccine stocks studied had its own characteristic profile of mutations that was identified by cDNA sequencing and quantitated by mutant analysis by PCR and restriction enzyme cleavage. Determination of the mutational profile of mumps vaccine lots could allow vaccine manufacturers to characterize seed viruses and monitor the consistency of vaccine production to prevent emergence of virulent revertants.