Multiple molecular pathways for fitness recovery of an RNA virus debilitated by operation of Muller's ratchet

Repeated bottleneck passages of RNA viruses result in fitness losses due to accumulation of deleterious mutations. We have analysed the molecular even ts underlying fitness recovery of a highly debilitated foot-and-mouth disea se virus (FMDV) clone, upon serial passage in BHK-21 cells. The debilitated clone included an unusual, internal polyadenylate extension preceding the second functional AUG initiation codon, and a number of additional mutation s scattered throughout the genome. Comparison of entire genomic nucleotide sequences in the course of passaging documented that loss of the internal p olyadenylate was the first event in the process of fitness recovery. Furthe r increases of fitness were associated with very few true reversions and wi th the accumulation of additional mutations affecting non-coding and coding regions. Remark ably, four biological subclones of the same debilitated FM DV clone gained fitness through three separate molecular pathways regarding correction of the internal polyadenylate: (i) a true reversion to yield th e wild-type sequence at the second functional AUG; (ii) a shortening of the internal polyadenylate tract; or (iii) a deletion of 69 residues spanning the site of the polyadenylate extension. The results document that an RNA v irus can find multiple pathways to reach alternative high fitness peaks on the fitness landscape. (C) 1999 Academic Press.