Discovery of Influenza A Virus Sequence Pairs and Their Combinations for Simultaneous Heterosubtypic Targeting that Hedge against Antiviral Resistance

The multiple circulating human influenza A virus subtypes coupled with the perpetual genomic mutations and segment reassortment events challenge the development of effective therapeutics. The capacity to drug most RNAs motivates the investigation on viral RNA targets. 123,060 segment sequences from 35,938 strains of the most prevalent subtypes also infecting humans–H1N1, 2009 pandemic H1N1, H3N2, H5N1 and H7N9, were used to identify 1,183 conserved RNA target sequences (≥15-mer) in the internal segments. 100% theoretical coverage in simultaneous heterosubtypic targeting is achieved by pairing specific sequences from the same segment (“Duals”) or from two segments (“Doubles”); 1,662 Duals and 28,463 Doubles identified. By combining specific Duals and/or Doubles to form a target graph wherein an edge connecting two vertices (target sequences) represents a Dual or Double, it is possible to hedge against antiviral resistance besides maintaining 100% heterosubtypic coverage. To evaluate the hedging potential, we define the hedge-factor as the minimum number of resistant target sequences that will render the graph to become resistant i.e. eliminate all the edges therein; a target sequence or a graph is considered resistant when it cannot achieve 100% heterosubtypic coverage. In an n-vertices graph (n ≥ 3), the hedge-factor is maximal (= n– 1) when it is a complete graph i.e. every distinct pair in a graph is either a Dual or Double. Computational analyses uncover an extensive number of complete graphs of different sizes. Monte Carlo simulations show that the mutation counts and time elapsed for a target graph to become resistant increase with the hedge-factor. Incidentally, target sequences which were reported to reduce virus titre in experiments are included in our target graphs. The identity of target sequence pairs for heterosubtypic targeting and their combinations for hedging antiviral resistance are useful toolkits to construct target graphs for different therapeutic objectives.Author Summary: An average of three influenza pandemics occurred in each century over the last 300 years. As occurrence of the next influenza pandemic is definite, developing new antivirals is imperative since resistance to the remaining class of antivirals has been reported occasionally, and vaccines are ineffective in the initial wave of a pandemic. The typical evolutionary traits of viruses, which manifest as multiple virus subtypes in circulation and perpetual viral genomic mutations, require the development of subtype-specific antivirals that ultimately acquire resistance. Being a rapidly evolving and highly contagious virus that manifest the most subtypes, this is particularly acute for influenza A. Our approach to overcome these challenges is to identify and characterize influenza A virus sequences for RNA targeting that can theoretically address all strains from the most prevalent human-infecting subtypes (i.e. simultaneous multi-subtype targeting) that can hedge against antiviral resistance. We uncover an extensive list of target sequence pairs and their specific combinations for which they can be selected for novel therapeutics development that will be effective on multiple circulating seasonal strains and future pandemic strains. As our approach is applicable to other viruses, the methods are general for use in the selection of antiviral therapeutic targets.