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The fidelity of flaviviruses is thought to be tightly regulated for optimal fitness within and between hosts. West Nile virus (WNV) high-fidelity (HiFi) mutations V793I and G806R within the RNA-dependent RNA polymerase, and low-fidelity (LoFi) mutation T248I within the methyltransferase, were previously shown to attenuate infectivity and replicative fitness in Culex mosquitoes and Culex tarsalis (CXT) cells but not in mammalian cells. We hypothesized that fidelity alterations would modify adaptation and maintenance in a host-specific manner. To test this hypothesis, wild-type (WT), HiFi (V793I/G806R) and LoFi (T248I) variants were sequentially passaged eight times in avian (PDE) or mosquito cells, or alternately between the two. Initial characterization confirmed that fidelity mutants are attenuated in mosquito, but not avian, cells. Deep sequencing revealed mutations unique to both cell lines and fidelity mutants, including ENV G1378A, a mutation associated with avian cell adaptation. To characterize maintenance and adaptation, viral outputs were monitored throughout passaging and viral fitness was assessed. The results indicate that fidelity mutants can at times recover fitness during mosquito cell passage, but remain attenuated relative to WT. Despite similar initial fitness, LoFi mutants were impaired during sequential passage in avian cells. Conversely, HiFi mutants passaged in avian cells showed increased adaptation, suggesting that increased fidelity may be advantageous in avian hosts. Although some adaptation occurred with individual mutants, the output titres of fidelity mutants were on average lower and were often lost during host switching. These data confirm that arbovirus fidelity is likely fine-tuned to maximize survival in disparate hosts.

Since 2015, the United States has experienced a resurgence in the number of mumps cases and outbreaks in fully vaccinated populations. These outbreaks have occurred predominantly in close-quarter settings, such as camps, colleges, and detention centers. Phylogenetic analysis of 758 mumps-positive samples from outbreaks across the United States identified 743 (98%) as genotype G based on sequence analysis of the mumps small hydrophobic (SH) gene. Additionally, SH sequences in the genotype G samples showed almost no sequence diversity, with 675 (91%) of them having identical sequences or only one nucleotide difference. This uniformity of circulating genotype and strain created complications for epidemiologic investigations and necessitated the development of a system for rapidly generating mumps whole-genome sequences for more detailed analysis. In this study, we report a novel and streamlined assay for whole-genome sequencing (WGS) of mumps virus genotype G. The WGS procedure successfully generated 318 high-quality WGS sequences on nucleic acid from genotype G-positive respiratory samples collected during several mumps outbreaks in the United States between 2016 and 2019. Sequencing was performed by a rapid and highly sensitive custom Ion AmpliSeq mumps genotype G panel, with sample preparation performed on an Ion Chef and sequencing on an Ion S5. The WGS data generated by the AmpliSeq panel provided enhanced genomic resolution for epidemiological outbreak investigations. Translation and protein sequence analysis also identified several potentially important epitope changes in the circulating mumps genotype G strains compared to the Jeryl-Lynn strain (JL5) used in vaccines in the United States, which could explain the current level of vaccine escapes.

Flaviviruses include several emerging and re-emerging arboviruses which cause millions of infections each year. Although relatively well-studied, much remains unknown regarding the mechanisms and means by which these viruses readily alternate and adapt to different hosts and environments. Here, we review a subset of the different aspects of flaviviral biology which impact host switching and viral fitness. These include the mechanism of replication and structural biology of the NS3 and NS5 proteins, which reproduce the viral genome; rates of mutation resulting from this replication and the role of mutational frequency in viral fitness; and the theory of quasispecies evolution and how it contributes to our understanding of genetic and phenotypic plasticity.