Molecular differences between two Jeryl Lynn mumps virus vaccine component strains, JL5 and JL2

The Jeryl Lynn (JL) vaccine against mumps virus (MuV) contains two components, MuVJL5 and MuVJL2, which differ by over 400 nt. Due to the occurrence of bias in the direction of mutation, these differences and those found in nucleotide sequences of different isolates of the minor component in the vaccine (MuVJL2) might be due to the effect of ADAR-like deaminases on MuV grown in tissue-cultured cells. A molecular clone of MuVJL2 (pMuVJL2) and MuVJL2-specific helper plasmids were constructed in order to investigate molecular interactions between MuVJL5 and MuVJL2, to augment the existing molecular clone of MuVJL5 (pMuVJL5) and MuVJL5-specific helper plasmids. Genome and mRNA termini of MuVJL2 were characterized, and an unusual oligo-G insertion transcriptional editing event was detected near the F mRNA polyadenylation site of MuVJL2, but not of MuVJL5. Genes encoding glycoproteins of rMuVJL2 and rMuVJL5 have been exchanged to characterize the oligo-G insertion, which associated with the specific sequence of the F gene of MuVJL2 and not with any other genes or the RNA-dependent RNA polymerase of strain MuVJL2. The results indicate that a single G-to-A sequence change obliterates the co-transcriptional editing of the F mRNA and that this oligo-G insertion does not affect the growth of the virus

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

Changes in Mumps Virus Gene Sequence Associated with Variability in Neurovirulent Phenotype

Mumps virus is highly neurotropic and, prior to widespread vaccination programs, was the major cause of viral meningitis in the United States. Nonetheless, the genetic basis of mumps virus neurotropism and neurovirulence was until recently not understood, largely due to the lack of an animal model. Here, nonneurovirulent (Jeryl Lynn vaccine) and highly neurovirulent (88-1961 wild type) mumps virus strains were passaged in human neural cells or in chicken fibroblast cells with the goal of neuroadapting or neuroattenuating the viruses, respectively. When tested in our rat neurovirulence assay against the respective parental strains, a Jeryl Lynn virus variant with an enhanced propensity for replication (neurotropism) and damage (neurovirulence) in the brain and an 88-1961 wild-type virus variant with decreased neurotropic and neurovirulent properties were recovered. To determine the molecular basis for the observed differences in neurovirulence and neuroattenuation, the complete genomes of the parental strains and their variants were fully sequenced. A comparison at the nucleotide level associated three amino acid changes with enhanced neurovirulence of the neuroadapted vaccine strain: one each in the nucleoprotein, matrix protein, and polymerase and three amino acid changes with reduced neurovirulence of the neuroattenuated wild-type strain: one each in the fusion protein, hemagglutinin-neuraminidase protein, and polymerase. The potential role of these amino acid changes in neurotropism, neurovirulence, and neuroattenuation is discussed

Preface

AcceptedThis is the author's version of the article. The final published version is available from the publisher via the link in this record.Harmonized regulation of clinical trials seems necessary in order to speed up approval processes and the time from bench to bedside for novel stem cell treatments. Europe is comprised of countries with diverse regulatory and health care systems and the regulators’ aim to unify practice across them provides an opportunity to study the effects of harmonization through regulation and novel institutions. We report findings from a long-term study alongside the first multinational European stem cell clinical trial that applied the harmonised approval procedures and follows the regulations in stem cell clinical trial practice standards introduced over the past decade. Adapting to this regulatory environment was costly in terms of trial implementation and caused delays in patient recruitment. That the stem cells for autologous use had to be transported all over Europe for processing in licensed centres stands out among the costly complications the teams had to address. We conclude that academic trials with little industry support are currently hampered by harmonised regulation. Such trials depend on complex cross-area expertise for implementation. The academic and public sectors have insufficient access to such information. In order to facilitate all promising routes toward a future stem cell medicine, a central support institution bundling expertise in handling the technicalities of implementing multinational trials in Europe is needed