Population Variability Generated during Rescue Process and Passaging of Recombinant Mumps Viruses

Figures

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  Figure 1. Partial scheme of pMRV2, showing positions of optimized T7 promoter (T7opt), hammerhead ribozyme (HHD-Rbz), complete mumps antigenomic sequence (in blue; consisting of leader sequence (Le), 7 genes (the nucleocapsid (N), phospho- (P), matrix (M), fusion (F), small hydrophobic (SH), hemagglutinin-neuraminidase (HN) and large (L) protein gene), intergenic regions and trailer sequence (Tr); not drawn strictly to scale) and hepatitis delta virus ribozyme (HDV-Rbz). Green and red lines indicate positions of differences to L-Zagreb vaccine consensus sequence. Dashed green lines indicate differences in noncoding regions (T1923C, A3175G, T6240A, A6241C and T8391G; the first letter indicates nucleotide identity in L-Zagreb vaccine consensus sequence) full green lines indicate synonymous substitutions (C8008T and C11176T), full red line indicates the nonsynonymous substitution (T9660C). The two insertion sites that were used in construction of plasmids with additional genomic segments are indicated.

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  Figure 2. Heterogenous genomic positions and variants’ frequencies in viral primary rescue stocks; the number of heterogenous positions is shown in each graph in the upper right corner. Blue and grey dashed lines emphasize 2% and 50%, respectively. Changes in viral consensus sequences (relative to input plasmid sequences) are specified above each graph (the first nucleotide/amino acid is the identity in plasmid); nucleotide change, protein and amino acid change are shown. The regions added to the mumps genome and the regions with alternative synonymous sequences are indicated below the x-axis in orange and green, respectively. The dots that indicate heterogeneities in those regions are also shown in orange or green.

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  Figure 3. Positions and percentages of six substitutions common to at least two primary viral rescue stocks. The changes indicate the difference from input plasmid sequence (the first nucleotide/amino acid is the identity in plasmid). The nucleotide change, gene and amino acid change are indicated, except for the synonymous substitution T1615C, where only the number of corresponding amino acid is shown, and for substitutions that occurred in noncoding region (NCR). For MRV3, F–RSV-MRV2, E1E2TMD-HCV-MRV2 and miscrMRV3 only the regions of mumps virus backbone (not the insert(s)) were included in the analysis, their genomic positions are adjusted to corresponding positions in MRV2.

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  Figure 4. Position of substitutions in viral consensus sequences relative to input plasmids. The number of substitutions is indicated in parenthesis next to viral sample name; no differences in consensus were found in MRV2. Only the regions of mumps virus backbone (not the inserts) were included in this analysis; the genomic positions for MRV3 and F–RSV–MRV2 were adjusted to corresponding positions in MRV2. Gene identities are shown above the horizontal line that represents the MuV antigenomic sequence in corresponding input plasmids.