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

ABSTRACT: 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.

SUBMITTER: Slovic A

PROVIDER: S-EPMC8707793 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In 2016, a year-long large-scale mumps outbreak occurred in Arkansas among a highly-vaccinated population. A total of 2954 mumps cases were identified during this outbreak. The majority of cases (1676 (57%)) were school-aged children (5-17 years), 1536 (92%) of these children had completed the mumps vaccination schedule. To weigh the possibility that the mumps virus evaded vaccine-induced immunity in the affected Arkansas population, we established a pipeline for genomic characterization of the outbreak strains. Our pipeline produces whole-genome sequences along with phylogenetic analysis of the outbreak mumps virus strains. We collected buccal swab samples of patients who tested positive for the mumps virus during the 2016 Arkansas outbreak, and used the portable Oxford Nanopore Technology to sequence the extracted strains. Our pipeline identified the genotype of the Arkansas mumps strains as genotype G and presented a genome-based phylogenetic tree with superior resolution to a standard small hydrophobic (SH) gene-based tree. We phylogenetically compared the Arkansas whole-genome sequences to all publicly available mumps strains. While these analyses show that the Arkansas mumps strains are evolutionarily distinct from the vaccine strains, we observed no correlation between vaccination history and phylogenetic grouping. Furthermore, we predicted potential B-cell epitopes encoded by the Arkansas mumps strains using a random forest prediction model trained on antibody-antigen protein structures. Over half of the predicted epitopes of the Jeryl-Lynn vaccine strains in the Hemagglutinin-Neuraminidase (HN) surface glycoprotein (a major target of neutralizing antibodies) region are missing in the Arkansas mumps strains. In-silico analyses of potential epitopes may indicate that the Arkansas mumps strains display antigens with reduced immunogenicity, which may contribute to reduced vaccine effectiveness. However, our in-silico findings should be assessed by robust experiments such as cross neutralization assays. Metadata analysis showed that vaccination history had no effect on the evolution of the Arkansas mumps strains during this outbreak. We conclude that the driving force behind the spread of the mumps virus in the 2016 Arkansas outbreak remains undetermined.

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

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