Figure 2. Experimental techniques to study viral evolutionary paths and aid in prediction.

A. A 2D genotype-fitness landscape underlying a hypothetical phenotypic transition (from purple to gold). B. Traversal of a viral population through such a landscape can be examined in a number of experimental ways. Phylogenetic reconstruction examines the evolutionary history of a viral population. Experimental evolution techniques measure the precise trajectories a given viral population takes to navigate a particular selective environment. Library-based screening allows further manipulation of the viral population, by exploring the local topography of the genotype-fitness landscape. C.) High resolution sequencing, such as Circular Sequencing (CirSeq), allows for the tracking of most single nucleotide mutations in a viral population, even at low frequencies. The error correction of CirSeq relies on the generation of head-to-tail cDNA repeats from circularized, fragmented RNA. Only mutations occurring in a majority of these repeats are identified as true mutations. If combined with serial passaging, such high resolution sequencing experiments can be used to estimate fitness effects across the genome and describe the evolutionary landscapes of viruses. D.) Deep mutational scanning (DMS) uses successive rounds of mutagenic PCR to generate a library of all possible codon or nucleotide substitutions of a given sequence. This library is then cloned back into the viral sequence for rescue and creation of mutant viruses. This population undergoes some form of selection (e.g. antibody selection), and the frequency of codon mutants postselection is determined by deep sequencing. A variety of methods can be used to determine amino acid preference at individual site across the gene of interest. Such preferences can be used to better inform phylogenetic reconstruction of viral evolution. E.) Microfluidics allow for massively parallel evolutionary experimentation and fine control of evolutionary and selective parameters influencing the evolution of viral populations. The ability to observe individual host cells, and individual virus particles and genomes, enables the quantification of important biological heterogeneity and dynamics underlying the infection process.