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Genetic stability of recombinant mumps viruses generated by reverse genetics technology

Reverse genetics technology enables recovery of infectious, replication-competent RNA virions from plasmids with cloned complementary DNA (cDNA). Among nonsegmented negative strand RNA viruses, viruses produced using reverse genetics have been based mostly on measles virus or vesicular stomatitis virus. The ability to manipulate mumps (MuV) genome by reverse genetics systems has been used as a tool for investigation of MuV biology and to develop MuV-based recombinant viruses with varying insert lengths (additional transcription units). In our work, we established a rescue system for MuV based on the consensus sequence of LZagreb vaccine. RNA viruses produced by rescue methodology are often referred to as viruses derived from infectious clone, implying that high genetic consistency of viral populations can be achieved. The goal of our research was to characterize the level of population diversity during the rescue processes ; seven different recombinant MuVs were rescued. The analysis of deep sequencing results showed that plasmids used in rescue are genetically homogenous, while viral populations in primal rescue stocks contain variants present mostly at low percentages. One substitution was observed in all 7 primary rescue stocks: C9660T leading to the amino acid change Pro408Leu in L protein. Interestingly, plasmid used in rescue codes for proline at this position (a mutation which was introduced during cloning), while original L- Zagreb vaccine strain, as well as all publicly available mumps sequences code for leucine, indicating this reversion could be important for function and/or structure of L protein.

mumps virus, reverse genetics, rescue, population diversity

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