gms | German Medical Science

Background: Influenza A virus (IAV), a major cause of morbidity and mortality, is continually evolving in response to all sorts of selective pressures. In addition to mutations that can lead to drug resistance, IAV evolves by reassortment. Using new sequencing methods we have begun to probe the nature of IAV evolution in response to selective pressures.

Methods: Our research uses a multidisciplinary team to rigorously probe the evolution of IAV under the impact of a variety of selective pressures from the environment. We use an integrated approach that combines viral passaging, novel deep sequencing methods, computational, and biophysical analysis to ascertain this impact. Using an in vitro evolution platform and whole-genome population sequencing, we investigated the population genomics of IAV during the reassortment of two strains or under the selective pressures of either oseltamivir (a licensed neuraminidase inhibitor) or favipiravir (an experimental antiviral thought to affect the viral polymerase). IAV was grown in MDCK cells under escalating concentrations of drug over serial passages. Samples were prepared using RNA purification, reverse transcription, whole genome PCR, followed by DNA barcoding and library preparation prior to sequencing on an Illumina HiSeq2000 platform.

Results: Selective pressure resulted in a variety of outcomes: when the two viral strains were simultaneously infecting cultured cells, reassortment and specific adaptation were reproducibly observed in multiple experiments. Surprisingly, specific mutations emerged exclusively with reassortants, suggesting possible mechanisms for why the reassortment occurs. Following treatment with oseltamivir, the known neuraminidase drug resistant H274Y mutation fixed reproducibly within the population without alterations of viral population diversity and persisted after withdrawal of the drug. In contrast, with the addition of high doses of favipiravir, sequence diversity markedly increased over time, with fixation of discrete mutants throughout the viral population. Saturation mutagenesis of defined portions of the IAV genome allows us to make predictions as to which mutations are likely to become fixed in the population as a result of selection.

Conclusion: Whole genome population sequencing is a powerful method for identifying viral genome responses to drugs, including identification of potential drug resistance mutations and reassortment-associated mutations. Such methods can be extended to viruses beyond influenza and with selective pressures including antibodies and host adaptation.