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

Recombinant mumps viruses (MuVs) based on established vaccine strains represent attractive vector candidates as they have known track records for high efficacy and the viral genome does not integrate in the host cells. We developed a rescue system based on the consensus sequence of the L- Zagreb vaccine and generated seven different recombinant MuVs by (a) insertion of one or two additional transcription units (ATUs), (b) lengthening of a noncoding region to the extent that the longest noncoding region in MuV genome is created, or (c) replacement of original L-Zagreb sequences with sequences rich in CG and AT dinucleotides. All viruses were successfully rescued and faithfully matched sequences of input plasmids. In primary rescued stocks, low percentages of heterogeneous positions were found (maximum 0.12%) and substitutions were predominantly obtained in minor variants, with maximally four substitutions seen in consensus. ATUs did not accumulate more mutations than the natural MuV genes. Six substitutions characteristic for recombinant viruses generated in our system were defined, as they repetitively occurred during rescue processes. In subsequent passaging of primary rescue stocks in Vero cells, different inconsistencies within quasispecies structures were observed. In order to assure that unwanted mutations did not emerge and accumulate, sub-consensus variability should be closely monitored. As we show for Pro408Leu mutation in L gene and a stop codon in one of ATUs, positively selected variants can rise to frequencies over 85% in only few passages

Influence of Ribavirin on Mumps Virus Population Diversity

Frequent mumps outbreaks in vaccinated populations and the occurrence of neurological complications (e.g., aseptic meningitis or encephalitis) in patients with mumps indicate the need for the development of more efficient vaccines as well as specific antiviral therapies. RNA viruses are genetically highly heterogeneous populations that exist on the edge of an error threshold, such that additional increases in mutational burden can lead to extinction of the virus population. Deliberate modulation of their natural mutation rate is being exploited as an antiviral strategy and a possibility for rational vaccine design. The aim of this study was to examine the ability of ribavirin, a broad-spectrum antiviral agent, to introduce mutations in the mumps virus (MuV) genome and to investigate if resistance develops during long-term in vitro exposure to ribavirin. An increase in MuV population heterogeneity in the presence of ribavirin has been observed after one passage in cell culture, as well as a bias toward C-to-U and G-to-A transitions, which have previously been defined as ribavirin-related. At higher ribavirin concentration, MuV loses its infectivity during serial passaging and does not recover. At low ribavirin concentration, serial passaging leads to a more significant increase in population diversity and a stronger bias towards ribavirin-related transitions, independently of viral strain or cell culture. In these conditions, the virus retains its initial growth capacity, without development of resistance at a whole-virus population level