Vaccines remain the most effective way of preventing infection and spread

Vaccines remain the most effective way of preventing infection and spread of infectious diseases. Also due to the high error rate of RNA viruses and selective pressures of the host environment these LAVs derived from such viruses can potentially revert back to wild-type virulence. This not only puts the vaccinee at risk but if shed can put those that are unvaccinated at risk as well. While these vaccines have been successful there still remains a need for a rational design strategy by which to create additional LAVs. One approach for rational vaccine design involves increasing the fidelity of the viral RdRp. Increased fidelity decreases the viral mutational frequency thereby reducing the genetic variation the virus needs in order to evade the host imposed bottlenecks to infection. While polymerase mutants exist which decrease viral mutation frequency the mutations are not in conserved regions of the polymerase which doesn’t lend itself toward using a common mutant approach toward developing a universal vaccine strategy for all RNA viruses. We have identified a conserved lysine residue in the active site of the PV RdRp that acts as a general acid during nucleotide incorporation. Mutation from a lysine to an arginine results in a high fidelity polymerase that replicates slowly thus creating an attenuated virus that is genetically stable and less likely to revert to a wild-type phenotype. This chapter provides detailed methods in which to identify the conserved lysine residue and evaluating fidelity and attenuation in cell culture (in vitro) and in the PV transgenic murine model (in vivo). [9–12]. Although a high mutation rate can lead to deleterious changes in the genome genetic diversity in RNA virus populations appears to be critical for fitness and survival and likely contributes to pathogenesis. In a heterogeneous pathogen population some variants are able to infect primary tissues and bypass host-imposed bottlenecks. From Irinotecan here the remaining variants can replicate into another heterogeneous population where some are once again able to bypass another layer of bottlenecks and perform secondary infection in other tissues thus demonstrating that a heterogeneous population or quasispecies can be beneficial to the pathogen. This adaptability poses a unique challenge for example when it comes to developing antiviral drugs and vaccines. RNA virus populations are heterogeneous due to error-prone replication by the viral RNA-dependent RNA polymerase (RdRp) which influences quasispecies evolution. This adaptability benefits the pathogen sometimes at the cost of the host. Currently error-prone replication is known to happen in all RNA viruses that infect both plants and CCND2 animals. It is also known that this error is due to rapid generation of variants and the fidelity of the viral RdRp [13–15]. In order to study the effect of polymerase mutants on RNA virus heterogeneity we turn to a model Irinotecan RNA virus poliovirus. Poliovirus (PV) belongs to the family TCA GCT ACA TTT GAA ACA-3′). Reverse: PV-3D-K359R-rev (5′-TGT TTC AAA TGT AGC TGA GTC AGC TGG AGT CAT-3′). T10E0.1 Buffer: 10 mM Tris–HCl pH 8.0 0.1 mM ethylene diamine tetraacetic acid [EDTA] pH 8.0. NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific). Deep Vent DNA polymerase 2000 U/mL (New England BioLabs). 3 mM dNTP mix: 100 mM dATP 100 mM dGTP 100 mM dTTP and 100 mM dCTP. Irinotecan This solution is prepared by combining 300 μL of each NTP and bringing the volume up to 10 mL with ultrapure water and can be aliquoted and stored indefinitely at 20 °C. 100 mM magnesium sulfate [MgSO4] solution (supplied with Deep Vent). 10 ThermoPol reaction buffer (supplied with Deep Vent). 3 M sodium acetate [NaOAc] pH 5.2: adjust pH with glacial acetic acid. Absolute ethanol. 70 % ethanol solution: 70 % EtOH 30 % ultrapure water. Omnipur Agarose (Millipore/Calbiochem). 0.5 TBE electrophoresis running buffer: 33 mM Tris–HCl 40 mM boric acid 1 mM EDTA pH 8.0 0.25 μg/mL ethidium Irinotecan bromide [EtBr]. Electrophoresis chamber and power source. 5 bromophenol blue [BPB]: 0.05 % bromophenol blue 50 % glycerol in T10E0.1 buffer. Tissue Culture Components). Complete medium (Tissue Culture Components). In vitro transcribed RNA made from mutant pMoVRA. VWR Signature Disposable Electroporation Cuvettes 2 mm (VWR). Bio-Rad Gene Pulser Generator Model 1652076 (Bio-Rad Laboratories). Bio-Rad Capacitance Extender Model 1652087 (Bio-Rad Laboratories). 2.3 Quantitating Virus and Viral Genomes Components HeLa S3 cells. Complete media (Tissue Culture Components)..