Expermental [i.e. Experimental] analysis of the mutation rate of influenza A viruses

Abstract

As with other ribonucleic acid (RNA) viruses, influenza A virus (IAV), with its error-prone RNA-dependent RNA polymerase (RdRP), is able to generate complex populations of genetically variant viruses referred to as quasispecies. Mutation-driven quasispecies diversity is the main mechanism for the acquisition of resistance to antivirals, immune escape, and host adaptations, which can impact pathogenesis and transmissibility. Current efforts in studying influenza mutation have been restricted to characterizing the functional roles of individual amino acids linked to influenza pathogenesis. A hitherto unexplored area of influenza research has been the role of quasispecies in the biology of influenza because there is limited methodology to accurately quantify influenza mutation rates and an absence of an unequivocal model to study influenza quasispecies dynamics. An accurate in vitro system was developed to assess the mutational frequencies and spectra of the RdRP of two IAV strains with different pathogenicity in mammals: human seasonal H3N2 A/Wuhan/359/95 (Wuhan) and highly pathogenic H5N1 A/Vietnam/1203/04 (VN1203). The intrinsic RdRP mutational frequencies and spectra of the two polymerase complexes were compared and validated using the spectra derived from live recombinant viruses. High-quality mutational spectra were generated to make possible the use of statistical and bioinformatics analysis. Statistically correlated mutational spectra and comparable mutational frequencies were observed, the first highly mutable motif of “AAAG” and “AAG” was discovered in both H5N1 and H3N2 RdRPs, as well as in viral progenies, which were recombinant A/Puerto Rico/8/34(PR8) viruses that differed in the RdRPs of the H3N2 and H5N1 strains. Non-segmented, positive-sense, single-stranded RNA viruses of the Piconaviridae family and the Togaviridae family with altered RdRP fidelity were selected in the presence of mutagens. To study the biological significance of the influenza mutation rate, ribavirin was demonstrated to mutagenize the IAV genome and was used to select mutagen-resistant variants of IAV that possess RdRP with altered fidelity. Through serial passages of A/Wuhan/359/95 virus in the presence of ribavirin in vitro, a mutation defective, high-fidelity (reduced mutation rate) IAV that exhibits decreased sensitivity to mutagens including ribavirin and manganese in vitro was generated and characterized. Using plaque reduction assay and mini-genome assay, the PB1-V43I mutation was identified to confer to decreased sensitivity to ribavirin and increased RdRP fidelity. Applying plasmid-based reverse genetics, the PB1-V43I mutation in the H3N2 and H5N1 viruses was shown to decrease mutation frequency by 1.0-3.1 folds. These mutation-defective viruses have decreased ability to generate monoclonal antibody escape mutants. Compared with wild-type, the mutation-defective H5N1 virus was shown to replicate to comparable titers in the lung of infected mice but is less lethal and has lower viral titers in the brain (not statistically significant). The first bioinformatics analysis of the influenza mutational spectra was conducted. By generating the first mutation-defective IAV, experimental data has shown that quasispecies diversity is crucial for IAV adaptation and pathogenicity. The discovery of high-fidelity IAV can provide an opportunity to investigate the molecular mechanisms leading to the generation of virus genetic diversity and to understand the role of quasispecies and population dynamics on influenza pathogenesis and transmission.