Viral Genome Dynamics During Antiviral Resistance Selection: A First Glimpse into Viral Evolution

The nature of the RNA-dependent RNA polymerase (RdRp: NS5B gene) of most RNA viruses renders their genome prone to the accumulation of mutations. Hence the virus population exists as a complex and dynamic mutant distribution i.e. a quasispecies. The quasispecies enables rapid adaptation of the virus to any changes in its natural environment. Also during antiviral therapy, rapid selection of drug resistant virus is facilitated by the existence of the virus as a quasispecies rather than a defined genomic sequence. We employed the bovine viral diarrhea virus (BVDV) [family of the Flaviviridae, genus pestivirus] as a model virus. Most, if not all of the currently known pestiviral RdRp inhibitors target a 7 A region between the amino acids F224 and E291 within the finger domain of the enzyme. Here we describe how the RdRp coding region of the viral genome evolves during in vitro antiviral resistance selection. In a first experiment, BVDV resistant to different selective inhibitors was independently selected using Method 1. In qualitative and phylogenetic analysis the sequence of drug-resistant strains, two clusters were observed (Fig 4 A, B, C). This observation might indicate that there exist certain genomic constraints that impede the potential truly random nature of the quasispecies. In order to test this hypothesis we chose two drugs for which the resistant genome sequence mapped to a different cluster (i.e. AG110 and BPIP). Furthermore we designed a resistance selection scheme (Method 2) that allowed monitoring of genomic changes in parallel and at intermediary passages. In the case that the selective pressure exerted by a specific compound class is constrained by certain genomic limitations, most of the sequences obtained during different passages would constantly cluster together and this in branches of the phylogenetic tree different from the other drug class(Fig 4 A, B, C). This would not be the case if BVDV has a truly random nature of a quasispecies. NUCLEOTIDES ALIGNMENT OF RESISTANT STRAINS