Maintenance of a host-specific minority mutation in the West Nile virus NS3.

West Nile virus (WNV), the most prevalent arthropod-borne virus (arbovirus) in the United States, is maintained in a cycle between Culex spp. mosquitoes and birds. Arboviruses exist within hosts and vectors as a diverse set of closely related genotypes. In theory, this genetic diversity can facilitate adaptation to distinct environments during host cycling, yet host-specific fitness of minority genotypes has not been assessed. Utilizing WNV deep-sequencing data, we previously identified a naturally occurring, mosquito-biased substitution, NS3 P319L. Using both cell culture and experimental infection in natural hosts, we demonstrated that this substitution confers attenuation in vertebrate hosts and increased transmissibility by mosquitoes. Biochemical assays demonstrated temperature-sensitive ATPase activity consistent with host-specific phenotypes. Together these data confirm the maintenance of host-specific minority variants in arbovirus mutant swarms, suggest a unique role for NS3 in viral fitness, and demonstrate that intrahost sequence data can inform mechanisms of host-specific adaptation.

The Role of the Flavivirus Replicase in Viral Diversity and Adaptation.

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.

A Chemical Strategy for Intracellular Arming of an Endogenous Broad-Spectrum Antiviral Nucleotide.

The naturally occurring nucleotide 3'-deoxy-3',4'-didehydro-cytidine-5'-triphosphate (ddhCTP) was recently found to exert potent and broad-spectrum antiviral activity. However, nucleoside 5'-triphosphates in general are not cell-permeable, which precludes the direct use of ddhCTP as a therapeutic. To harness the therapeutic potential of this endogenous antiviral nucleotide, we synthesized phosphoramidate prodrug HLB-0532247 (1) and found it to result in dramatically elevated levels of ddhCTP in cells. We compared 1 and 3'-deoxy-3',4'-didehydro-cytidine (ddhC) and found that 1 more effectively reduces titers of Zika and West Nile viruses in cell culture with minimal nonspecific toxicity to host cells. We conclude that 1 is a promising antiviral agent based on a novel strategy of facilitating elevated levels of the endogenous ddhCTP antiviral nucleotide.

Divergent Mutational Landscapes of Consensus and Minority Genotypes of West Nile Virus Demonstrate Host and Gene-Specific Evolutionary Pressures.

Our current understanding of the natural evolution of RNA viruses comes largely from consensus level genetic analyses which ignore the diverse mutant swarms that comprise within-host viral populations. The breadth and composition of viral mutant swarms impact viral fitness and adaptation, and the capacity for swarm plasticity is likely to be particularly important for arthropod-borne viruses (arboviruses) that cycle between taxonomically divergent hosts. Despite this, characterization of the relationship between the selective pressures and genetic signatures of the mutant swarm and consensus sequences is lacking. To clarify this, we analyzed previously generated whole genome, deep-sequencing data from 548 West Nile virus samples isolated from avian tissues or mosquitoes in New York State from 1999-2018. Both consensus level (interhost) and minority level (intrahost) nucleotide and amino acid sequences were analyzed, and diversity at each position was calculated across the genome in order to assess the relationship between minority and consensus sequences for individual genes and hosts. Our results indicate that consensus sequences are an inept representation of the overall genetic diversity. Unique host and gene-specific signatures and selective pressures were identified. These data demonstrate that an accurate and comprehensive understanding of arbovirus evolution and adaptation within and between hosts requires consideration of minority genotypes.

West Nile Virus fidelity modulates the capacity for host cycling and adaptation.

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.